Methods and compositions for amplification of RNA sequences

ABSTRACT

The invention provides methods for linear and exponential amplification of RNA. They are particularly suitable for amplifying a plurality of RNA species in a sample. The methods are based on hybridization of polynucleotide comprising a propromoter sequence to a primer extension product to generate an intermediate polynucleotide capable of driving transcription, whereby multiple copies of RNA products comprising sequences complementary to an RNA sequence of interest are generated. The methods are useful for preparation of nucleic acid libraries and substrates for analysis of gene expression of cells in biological samples. The invention also provides compositions and kits for practicing the amplification methods, as well as methods which use the amplification products.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of the provisional patentapplication U.S. Ser. No. 60/274,236, filed Mar. 9, 2001, which isincorporated by reference in its entirety.

TECHNICAL FIELD

The invention relates to the field of polynucleotide amplification. Moreparticularly, the invention provides methods, compositions and kits foramplifying (i.e., making multiple copies) RNA sequences of interestwhich employ a polynucleotide comprising a propromoter and RNAtranscription.

BACKGROUND ART

The ability to amplify ribonucleic acid (RNA) is an important aspect ofefforts to elucidate biological processes. To date, RNA (generally,mRNA) amplification is most commonly performed using the reversetranscriptase-polymerase chain reaction (RT-PCR) method and variationsthereof. These methods are based on replication of RNA by reversetranscriptase to form single stranded DNA complementary to the RNA(cDNA), which is followed by polymerase chain reaction (PCR)amplification to produce multiple copies of double stranded DNA.Although these methods are most commonly used, they have somesignificant drawbacks: a) the reactions require thermocycling; b) theproducts are double stranded, thus rendering them less accessible tobinding to probes; c) the reactions are prone to contamination withproducts of prior amplification, thus requiring strict containment ofreaction mixtures; and d) the exponential nature of amplification ofthese methods renders them prone to generate pools of products which donot truly reflect the representation of the various RNA sequences in theinput total RNA sample, due to unequal efficiency of amplification ofdifferent sequences, and the nature of exponential amplification whichis based on replication of amplification products rather than oncontinued replication of the input target RNAs.

The total cellular mRNA represents gene expression activity at a definedtime. Gene expression is affected by cell cycle progression,developmental regulation, response to internal and external stimuli andthe like. The profile of expressed genes for any cell type in anorganism reflects normal or disease states, response to various stimuli,developmental stages, cell differentiation, and the like.

Various methods for the analysis of gene expression have be developed inrecent years. See, for example, U.S. Pat. Nos. 5,744,308; 6,143,495;5,824,517; 5,829,547; 5,888,779; 5,545,522; 5,716,785; 5,409,818; EP0971039A2; EP 0878553A2. These include quantification of specific mRNAs,and the simultaneous quantification of a large number of mRNAs, as wellas the detection and quantification of patterns of expression of knownand unknown genes. The analysis of gene expression profiles is currentlyone of the most powerful tools in the study of cellular differentiationand cellular development, and in the investigation of normal and diseasestates of various organisms, in particular in human. This analysis iscrucial for gene discovery, molecular medicine and drug discoveryprocesses.

Essential for gene expression profiling is the ability to randomlyamplify the total cellular mRNAs prepared from any cell or tissue.Although analysis of non-amplified mRNA is feasible, a significantamount of starting mRNA would be required. However, the total amount ofsample mRNA that is available is frequently limited by the amount ofbiological sample from which it is derived. Biological samples are oftenlimited in amount and precious. Moreover, the amount of the various mRNAspecies is not equal; some species are more abundant than others, andthese are more likely and easier, to analyze. The ability to amplifymRNA sequences enables the analysis of less abundant, rare mRNA species.The ability to analyze small samples, by means of nucleic acidamplification, is also advantageous for design parameters of large scalescreening of effector molecule libraries, for which reduction in samplevolume is a major concern both for the ability to perform very largescale screening or ultra high throughput screening, and in view of thelimiting amounts of library components.

Therefore, there is a need for improved RNA amplification methods thatovercome drawbacks in existing methods. The invention provided hereinfulfills this need and provides additional benefits.

All references cited herein, including patent applications andpublications, are incorporated by reference in their entirety.

DISCLOSURE OF THE INVENTION

The invention provides methods, compositions, and kits forpolynucleotide, specifically ribonucleic acid, amplification, as well asapplications of the amplification methods.

In one aspect, the invention provides methods of generating multiplecopies of the complementary sequence of an RNA sequence of interest,said method comprising the steps of: (a) extending a first primerhybridized to a target RNA with an RNA-dependent DNA polymerase, wherebya complex comprising a first primer extension product and the target RNAis produced; (b) cleaving RNA in the complex of step (b) with an enzymethat cleaves RNA from an RNA/DNA hybrid; (c) extending a second primerhybridized to the first primer extension product with a DNA-dependentDNA polymerase, whereby a complex comprising the first primer extensionproduct and a second primer extension product is produced; (d)denaturing the complex of step (c); and (e) hybridizing to the secondprimer extension product a propromoter polynucleotide comprising apropromoter and a region which hybridizes to the second primer extensionproduct under conditions which allow transcription to occur by RNApolymerase, such that RNA transcripts are produced comprising sequencescomplementary to the target RNA; whereby multiple copies of thecomplementary sequence of the RNA sequence of interest are generated.

In one aspect, the invention provides methods of generating multiplecopies of (amplifying) the complementary sequence of an RNA sequence ofinterest, said method comprising the steps of: (a) hybridizing a firstprimer to a target ribonucleic acid; (b) extending the first primer withan RNA-dependent DNA polymerase, whereby a complex comprising a firstprimer extension product and the target ribonucleic acid is produced;(c) cleaving ribonucleic acid in the complex of step (b) with an enzymethat cleaves RNA from an RNA/DNA hybrid; (d) hybridizing a second primerto the first primer extension product; (e) extending the second primerwith a DNA-dependent DNA polymerase, whereby a complex comprising thefirst primer extension product and a second primer extension product isproduced; (f) denaturing the complex of step (e); (g) hybridizing to thesecond primer extension product a polynucleotide comprising apropromoter and a region which hybridizes to the second primer extensionproduct under conditions which allow transcription to occur by RNApolymerase, such that RNA transcripts are produced comprising sequencescomplementary to the target ribonucleic acid, whereby multiple copies ofthe complementary sequence of the RNA sequence of interest aregenerated. In some embodiments, the polynucleotide comprising apropromoter is a propromoter template oligonucleotide (PTO). In someembodiments, the invention provides methods of generating multiplecopies of the complementary sequence of an RNA sequence of interest,said methods comprising the steps of: (a) combining: a single strandedsecond primer extension product resulting from step (f) of the aspect ofthe invention described above; a propromoter polynucleotide comprising apropromoter and a region which is hybridizable to a single strandedsecond primer extension product; and an RNA polymerase; and (b)incubating the mixture of step (a) under conditions (which includesnecessary substrates and buffer conditions) that permit propromoterpolynucleotide hybridization and RNA transcription, whereby multiplecopies of the complementary sequence of the RNA sequence of interest aregenerated. It is understood that any combination of these incubationsteps, and any single incubation step, to the extent that the incubationis performed as part of any of the methods described herein, fall withinthe scope of the invention. It is also understood that methods thatcomprise one or more incubation steps do not require a separatecombination step, as such combinations are implicit in incubating thereaction mixture(s).

In another aspect, the invention provides methods of generating multiplecopies of the complementary sequence of an RNA sequence of interest,said method comprising the steps of: (a) extending a first primerhybridized to a target RNA with an RNA-dependent DNA polymerase, wherebya complex comprising a first primer extension product and the target RNAis produced; (b) cleaving RNA in the complex of step (a) with an enzymethat cleaves RNA from an RNA/DNA hybrid; (c) extending a second primerhybridized to the first primer extension product with a DNA-dependentDNA polymerase, whereby a complex comprising the first primer extensionproduct and a second primer extension product is produced; (d)denaturing the complex of step (c); (e) hybridizing to the second primerextension product a propromoter polynucleotide comprising a propromoterand a region which hybridizes to the second primer extension productunder conditions which allow transcription to occur by RNA polymerase,such that RNA transcripts are produced comprising sequencescomplementary to the target RNA; (f) extending a third primer hybridizedto said RNA transcripts with an RNA-dependent DNA polymerase, whereby acomplex comprising a third primer extension product and an RNAtranscript is produced; (g) cleaving RNA in the complex of step (f) withan enzyme that cleaves RNA from an RNA/DNA hybrid; (h) hybridizing apropromoter polynucleotide comprising a propromoter and a region whichhybridizes to a single stranded third primer extension product underconditions which allow transcription to occur by RNA polymerase, suchthat RNA transcripts are produced comprising sequences complementary tothe target RNA; (i) optionally repeating steps (f) to (h); wherebymultiple copies of the complementary sequence of the RNA sequence ofinterest are produced.

In another aspect, the invention provides methods of generating multiplecopies of (amplifying) the complementary sequence of an RNA sequence ofinterest, said method comprising the steps of: (a) hybridizing a firstprimer to a target ribonucleic acid; (b) extending the first primer withan RNA-dependent DNA polymerase, whereby a complex comprising a firstprimer extension product and the target ribonucleic acid is produced;(c) cleaving ribonucleic acid in the complex of step (b) with an enzymethat cleaves RNA from an RNA/DNA hybrid; (d) hybridizing a second primerto the first primer extension product; (e) extending the second primerwith a DNA-dependent DNA polymerase, whereby a complex comprising thefirst primer extension product and a second primer extension product isproduced; (f) denaturing the complex of step (e); (g) hybridizing to thesecond primer extension product a propromoter and a region whichhybridizes to the second primer extension product under conditions whichallow transcription to occur by RNA polymerase, such that RNAtranscripts are produced comprising sequences complementary to thetarget ribonucleic acid; (h) hybridizing a third primer to said RNAtranscripts; (i) extending the third primer with an RNA-dependent DNApolymerase, whereby a complex comprising a third primer extensionproduct and an RNA transcript is produced; (j) cleaving RNA in thecomplex of step (i) with an enzyme that cleaves RNA from an RNA/DNAhybrid; (k) hybridizing a propromoter polynucleotide comprising apropromoter and a region which hybridizes to a single stranded thirdprimer extension product under conditions which allow transcription tooccur by RNA polymerase, such that RNA transcripts are producedcomprising sequences complementary to the target ribonucleic acid; (l)optionally repeating steps (h) to (k), whereby multiple copies of thecomplementary sequence of the RNA sequence of interest are produced. Insome embodiments, the polynucleotide comprising a propromoter is apropromoter template oligonucleotide (PTO). In some embodiments, theinvention provides methods of generating multiple copies of thecomplementary sequence of an RNA sequence of interest, said methodscomprising the steps of: (a) combining: a single stranded second primerextension product resulting from step (f) described above in thisparagraph; a third primer comprising a sequence hybridizable to an RNAtranscript comprising a sequence complementary to the target RNA; apropromoter polynucleotide comprising a propromoter and a region whichis hybridizable to a single stranded second primer extension product; apropromoter polynucleotide comprising a propromoter and a region whichis hybridizable to a single stranded third primer extension product; anenzyme that cleaves RNA from an RNA/DNA hybrid; and an RNA polymerase;and (b) incubating the mixture of step (a) under conditions (whichincludes necessary substrates and buffers) that permit primer extension,RNA cleavage, propromoter polynucleotide hybridization and RNAtranscription, whereby multiple copies of the complementary sequence ofthe RNA sequence of interest are generated. In yet another embodiment,the invention provides methods of generating multiple copies of thecomplementary sequence of an RNA sequence of interest, said methodscomprising the steps of: (a) combining: an RNA transcript from step (g)described above in this paragraph; a third primer comprising a sequencehybridizable to the RNA transcript; a propromoter polynucleotidecomprising a propromoter and a region which is hybridizable to a singlestranded third primer extension product; an enzyme that cleaves RNA froman RNA/DNA hybrid; and an RNA polymerase; and (b) incubating the mixtureof step (a) under conditions (which includes necessary substrates andbuffers) that permit primer extension, RNA cleavage, propromoterpolynucleotide hybridization and RNA transcription, whereby multiplecopies of the complementary sequence of the RNA sequence of interest aregenerated.

In still another aspect, the invention provides methods of generatingmultiple copies of (amplifying) the complementary sequence of an RNAsequence of interest, said method comprising the steps of: (a)combining: a target ribonucleic acid; a first primer comprising asequence that is hybridizable to the target ribonucleic acid; a secondprimer comprising a sequence hybridizable to an extension product of thefirst primer; a propromoter polynucleotide comprising a propromoter anda region which is hybridizable to a single stranded second primerextension product; an RNA-dependent DNA polymerase; a DNA-dependent DNApolymerase; an RNA polymerase; and an enzyme that cleaves RNA from anRNA/DNA hybrid; and (b) incubating the mixture of step (a) underconditions (which includes necessary substrates and buffer conditions)that permit primer hybridization, primer extension, RNA cleavage,propromoter polynucleotide hybridization, and RNA transcription. In someembodiments, the polynucleotide comprising a propromoter is apropromoter template oligonucleotide (PTO).

In yet another aspect, the invention provides methods of generatingmultiple copies of (amplifying) the complementary sequence of an RNAsequence of interest, said method comprising the steps of: (a)combining: a target ribonucleic acid; a first primer comprising asequence that is hybridizable to the target ribonucleic acid; a secondprimer comprising a sequence hybridizable to an extension product of thefirst primer; a third primer comprising a sequence hybridizable to anRNA transcript comprising a sequence complementary to the targetribonucleic acid; a propromoter polynucleotide comprising a propromoterand a region which is hybridizable to a single stranded second primerextension product; a propromoter polynucleotide comprising a propromoterand a region which is hybridizable to a single stranded third primerextension product; an RNA-dependent DNA polymerase; a DNA-dependent DNApolymerase; an RNA polymerase; and an enzyme that cleaves RNA from anRNA/DNA hybrid; and (b) incubating the mixture of step (a) underconditions (which includes necessary substrates and buffer conditions)that permit primer hybridization, primer extension, RNA cleavage,propromoter polynucleotide hybridization, and RNA transcription. In someembodiments, the polynucleotide comprising a propromoter is apropromoter template oligonucleotide (PTO).

In another aspect, the invention provides methods of generating multiplecopies of the complementary sequence of an RNA sequence of interest,said method comprising the steps of: (a) extending a first primerhybridized to a target RNA with an RNA-dependent DNA polymerase, wherebya complex comprising a first primer extension product and the target RNAis produced; (b) cleaving RNA in the complex of step (b) with an enzymethat cleaves RNA from an RNA/DNA hybrid; (c) extending a second primerhybridized to the first primer extension product with a DNA-dependentDNA polymerase, whereby a complex comprising the first primer extensionproduct and a second primer extension product is produced; (d)denaturing the complex of step (c); and (e) hybridizing to the secondprimer extension product a propromoter polynucleotide comprising apropromoter and a region which hybridizes to the second primer extensionproduct under conditions which allow transcription to occur by RNApolymerase, such that RNA transcripts are produced comprising sequencescomplementary to the target RNA; whereby multiple copies of thecomplementary sequence of the RNA sequence of interest are generated.

In another aspect, the invention provides methods of generating multiplecopies of the complementary sequence of an RNA sequence of interest,said method comprising the steps of: (a) extending a first primerhybridized to a target RNA with an RNA-dependent DNA polymerase, wherebya complex comprising a first primer extension product and the target RNAis produced; (b) cleaving RNA in the complex of step (a) with an enzymethat cleaves RNA from an RNA/DNA hybrid; (c) extending a second primerhybridized to the first primer extension product with a DNA-dependentDNA polymerase, whereby a complex comprising the first primer extensionproduct and a second primer extension product is produced; (d)denaturing the complex of step (c); (e) hybridizing to the second primerextension product a propromoter polynucleotide comprising a propromoterand a region which hybridizes to the second primer extension productunder conditions which allow transcription to occur by RNA polymerase,such that RNA transcripts are produced comprising sequencescomplementary to the target RNA; (f) extending a third primer hybridizedto said RNA transcripts with an RNA-dependent DNA polymerase, whereby acomplex comprising a third primer extension product and an RNAtranscript is produced; (g) cleaving RNA in the complex of step (f) withan enzyme that cleaves RNA from an RNA/DNA hybrid; (h) hybridizing apropromoter polynucleotide comprising a propromoter and a region whichhybridizes to a single stranded third primer extension product underconditions which allow transcription to occur by RNA polymerase, suchthat RNA transcripts are produced comprising sequences complementary tothe target RNA; (i) optionally repeating steps (f) to (h); wherebymultiple copies of the complementary sequence of the RNA sequence ofinterest are produced.

In another aspect, the invention provides a method of generatingmultiple copies of the complementary sequence of an RNA sequence ofinterest comprising incubating a reaction mixture, said reaction mixturecomprising: (a) a single stranded second primer extension productresulting from step (d) above; (b) a propromoter polynucleotidecomprising a propromoter and a region which is hybridizable to a singlestranded second primer extension product; and an RNA polymerase; whereinthe incubation is under conditions that permit propromoterpolynucleotide hybridization and RNA transcription, whereby multiplecopies of the complementary sequence of the RNA sequence of interest aregenerated.

In another aspect, the invention provides methods of generating multiplecopies of the complementary sequence of an RNA sequence of interestcomprising incubating a reaction mixture, said reaction mixturecomprising: (a) a single stranded second primer extension productresulting from step (d) above; (b) a third primer comprising a sequencehybridizable to an RNA transcript comprising a sequence complementary tothe target RNA; (c) a propromoter polynucleotide comprising apropromoter and a region which is hybridizable to a single strandedsecond primer extension product; (d) a propromoter polynucleotidecomprising a propromoter and a region which is hybridizable to a singlestranded third primer extension product; (e) an enzyme that cleaves RNAfrom an RNA/DNA hybrid; and (f) an RNA polymerase; wherein theincubation is under conditions that permit primer extension, RNAcleavage, propromoter polynucleotide hybridization and RNAtranscription, whereby multiple copies of the complementary sequence ofthe RNA sequence of interest are generated.

In another aspect, the invention provides methods of generating multiplecopies of the complementary sequence of an RNA sequence of interest,said method comprising incubating a reaction mixture, said reactionmixture comprising: (a) an RNA transcript from step (e) above, (b) athird primer comprising a sequence hybridizable to the RNA transcript;(c) a propromoter polynucleotide comprising a propromoter and a regionwhich is hybridizable to a single stranded third primer extensionproduct; (d) an enzyme that cleaves RNA from an RNA/DNA hybrid; and (e)an RNA polymerase; wherein the incubation is under conditions thatpermit primer extension, RNA cleavage, propromoter polynucleotidehybridization and RNA transcription, whereby multiple copies of thecomplementary sequence of the RNA sequence of interest are generated.

In another aspect, the invention provides methods of generating multiplecopies of the complementary sequence of an RNA sequence of interest,said method comprising incubating a reaction mixture, said reactionmixture comprising: (a) a target RNA; (b) a first primer comprising asequence that is hybridizable to the target RNA; (c) a second primercomprising a sequence hybridizable to an extension product of the firstprimer; (d) a propromoter polynucleotide comprising a propromoter and aregion which is hybridizable to a single stranded second primerextension product; (e) an RNA-dependent DNA polymerase; (f) aDNA-dependent DNA polymerase; (g) an RNA polymerase; and (h) an enzymethat cleaves RNA from an RNA/DNA hybrid; wherein the incubation is underconditions that permit primer hybridization, primer extension, RNAcleavage, propromoter polynucleotide hybridization, and RNAtranscription, whereby multiple copies of the complementary sequence ofthe RNA sequence of interest are generated.

In another aspect, invention provides methods of generating multiplecopies of the complementary sequence of an RNA sequence of interest,said method comprising incubating a reaction mixture, said reactionmixture comprising: (a) a target RNA; (b) a first primer comprising asequence that is hybridizable to the target RNA; (c) a second primercomprising a sequence hybridizable to an extension product of the firstprimer; (d) a third primer comprising a sequence hybridizable to an RNAtranscript comprising a sequence complementary to the target RNA; (e) apropromoter polynucleotide comprising a propromoter and a region whichis hybridizable to a single stranded second primer extension product;(f) a propromoter polynucleotide comprising a propromoter and a regionwhich is hybridizable to a single stranded third primer extensionproduct; (g) an RNA-dependent DNA polymerase;(h) a DNA-dependent DNApolymerase; (i) an RNA polymerase; and (j) an enzyme that cleaves RNAfrom an RNA/DNA hybrid; wherein the incubation is conditions that permitprimer hybridization, primer extension, RNA cleavage, propromoterpolynucleotide hybridization, and RNA transcription, whereby multiplecopies of the complementary sequence of the RNA sequence of interest aregenerated.

In another aspect, the invention provides methods of generating multiplecopies of the complementary sequence of an RNA sequence of interest,said method comprising: (a) hybridizing a composite primer to a singlestranded second primer extension product resulting from step (d) above,wherein the composite primer comprises an RNA portion and a 3′ DNAportion; (b) extending the composite primer with a DNA-dependent DNApolymerase, whereby a complex comprising a primer extension product andthe second primer extension product is formed; (c) cleaving RNA in thecomplex of step (b) with an enzyme that cleaves RNA from an RNA/DNAhybrid, such that another composite primer hybridizes to the secondprimer extension product and repeats primer extension by stranddisplacement, whereby multiple copies of the complement of the RNAsequence of interest are produced.

In another aspect, the invention provides methods of generating multiplecopies of a polynucleotide sequence complementary to an RNA sequence ofinterest, said method comprising the steps of: (a) extending a compositeprimer hybridized to a second primer extension product, wherein saidprimer extension product comprises a complement of a first primerextension product generated by extension of a first primer hybridized totemplate RNA by any of the methods described herein; whereby said firstprimer extension product is displaced, and whereby multiple copies of apolynucleotide sequence complementary to the RNA sequence of interestare generated.

In another aspect, the invention provides methods of generating multiplecopies of the complementary sequence of an RNA sequence of interest,said method comprising incubating a reaction mixture, said reactionmixture comprising: (a) a single stranded second primer extensionproduct resulting from step (d) above; (b) a composite primer which ishybridizable to the single stranded second primer extension product,wherein the composite primer comprises an RNA portion and a 3′ DNAportion; (c) DNA-dependent DNA polymerase; (d) an enzyme that cleavesRNA from an RNA/DNA hybrid; wherein the incubation is made underconditions that permit composite primer hybridization, RNA cleavage, anddisplacement of the primer extension product from the complex of step(a) described above when its RNA is cleaved and a composite primer bindsto the primer extension product in the complex, whereby multiple copiesof the complement of the RNA sequence of interest are produced.

In another aspect, the invention provides methods of generating multiplecopies of an RNA sequence of interest, said method comprising the stepsof: (a) extending a composite primer hybridized to a second primerextension product, wherein said primer extension product comprises acomplement of a first primer extension product generated by extension ofa first primer hybridized to template RNA by any of the methodsdescribed herein; whereby said first primer extension product isdisplaced, (b) hybridizing the displaced first primer extension productwith a polynucleotide comprising a propromoter and a region which ishybridizable to the displaced first primer extension product underconditions which allow transcription to occur by RNA polymerase, suchthat RNA transcripts are produced comprising sequences complementary tothe displaced primer extension products, whereby multiple copies of theRNA sequence of interest are generated.

As is clear to one skilled in the art, reference to production of copiesof an RNA or DNA sequence of interest or copies of a polynucleotidesequence complementary to an RNA or DNA sequence of interest refers toproducts that may contain, comprise or consist of such sequences. As isevident to one skilled in the art, aspects that refer to combining andincubating the resultant mixture also encompasses method embodimentswhich comprise incubating the various mixtures (in various combinationsand/or subcombinations) so that the desired products are formed. It isunderstood that any combination of these incubation steps, and anysingle incubation step, to the extent that the incubation is performedas part of any of the methods described herein, fall within the scope ofthe invention. It is also understood that methods that comprise one ormore incubation steps do not require a separate combination step, assuch combinations are implicit in incubating the reaction mixture(s)

Various embodiments of the primers are used in the methods of theinvention. For example, in some embodiments, the first primer comprisesa 5′ portion that is not hybridizable (under a given set of conditions)to a target ribonucleic acid. In some of these embodiments, the 5′portion comprises a sequence the complement of which is hybridizable bya propromoter polynucleotide under a given set of conditions. In oneexample, the presence of said 5′ portion in the first primer results ingeneration of a second primer extension product that is hybridizable(under a given set of conditions) by a propromoter polynucleotide. Inanother example, the presence of said 5′ portion in the first primerresults in generation of a third primer extension product that ishybridizable (under a given set of conditions) by a propromoterpolynucleotide. In some embodiments wherein a target RNA is mRNA, thefirst primer may comprise a poly-T sequence. In other embodiments, thesecond primer and the third primer are the same. In still anotherembodiment, the second primer and the third primer are different. In yetanother embodiment, the second primer and the third primer hybridize todifferent complementary sequences. In some embodiments, the secondand/or third primer comprises a sequence (for example, a 3′ sequence)that is a random sequence. In yet other embodiments, the second and/orthird primer is a random primer. In yet other embodiments, the thirdprimer is a composite primer. In some embodiments, the RNA portion of acomposite primer is 5′ with respect to the 3′ DNA portion. In stillother embodiments, the 5′ RNA portion is adjacent to the 3′ DNA portion.In other embodiments, the composite primer comprises the sequence of afirst primer.

The enzymes which may be used in the methods and compositions aredescribed herein. For example, the enzyme that cleaves RNA may be anRNase H, and the RNA-dependent DNA polymerase may be reversetranscriptase. The RNA-dependent DNA polymerase may comprise an RNase Henzyme activity. Similarly, a DNA polymerase may comprise bothRNA-dependent and DNA-dependent DNA polymerase enzyme activities. ADNA-dependent DNA polymerase and an enzyme that cleaves RNA may also bethe same enzyme. A DNA-dependent DNA polymerase, an RNA-dependent DNApolymerase, and the enzyme that cleaves RNA can also be the same enzyme.

In some embodiments, methods of the invention are used to generatelabeled polynucleotide products (generally DNA or RNA products). In someembodiments of methods for generating labeled DNA products, at least onetype of dNTP used is a labeled dNTP. In some embodiments of methods forgenerating labeled RNA products, at least one type of rNTP used is alabeled rNTP. In other embodiments of methods for generating labeled DNAproducts, a labeled composite primer is used.

In some embodiments, the methods of the invention employ a propromoterpolynucleotide (for example, a PTO) that comprises a region at the 3′end which hybridizes to the second or third primer extension products,whereby DNA polymerase extension of the extension products produces adouble stranded promoter from which transcription occurs.

The methods are applicable to amplifying any RNA target, including, forexample, mRNA and ribosomal RNA. One or more steps may be combinedand/or performed sequentially (often in any order, as long as therequisite product(s) are able to be formed). It is also evident, and isdescribed herein, that the invention encompasses methods in which theinitial, or first, step is any of the steps described herein. Forexample, the methods of the invention do not require that the first stepbe production of the first primer extension product from the RNAtemplate. Methods of the invention encompass embodiments in which later,“downstream” steps are an initial step.

The invention also provides methods which employ (usually, analyze) theproducts of the amplification methods of the invention, such assequencing, detection of sequence alteration(s) (e.g., genotyping ornucleic acid mutation detection); determining presence or absence of asequence of interest; gene expression profiling; subtractivehybridization; preparation of a subtractive hybridization probe;differential amplification; preparation of libraries (including cDNA anddifferential expression libraries); preparation of an immobilizednucleic acid (which can be a nucleic acid immobilized on a microarray),and characterizing (including detecting and/or quantifying) amplifiednucleic acid products generated by the methods of the invention.

In one aspect, the invention provides methods of sequencing an RNAsequence of interest, said method comprising (a) amplifying a targetribonucleic acid containing the sequence of interest by the methodsdescribed herein in the presence of a mixture of rNTPs and rNTP analogssuch that transcription is terminated upon incorporation of an rNTPanalog; and (b) analyzing the amplification products to determinesequence.

In another aspect, the invention provides methods of sequencing an RNAsequence of interest, said method comprising (a) amplifying a targetribonucleic acid containing the sequence of interest by the methodsdescribed herein, wherein RNA transcripts generated from the secondprimer extension product are amplified in the presence of a mixture ofrNTPs and rNTP analogs such that transcription is terminated uponincorporation of an rNTP analog; and (b) analyzing the amplificationproducts to determine sequence.

In some aspects, the invention provides methods of sequencing an RNAsequence of interest, said methods comprising amplifying a target RNAcontaining the sequence of interest by the amplification methods of theinvention in the presence of a mixture of dNTPs and dNTP analogs (whichmay be labeled or unlabeled), such that primer extension is terminatedupon incorporation of a dNTP analog which may be labeled or unlabeled,and analyzing the amplification products to determine sequence.

In some aspects, the invention provides methods of detecting a mutation(or, in some aspects, characterizing a sequence) in a target ribonucleicacid, comprising (a) amplifying the target ribonucleic acid by a methoddescribed herein; and (b) analyzing the amplification products of themethod for single stranded conformation, wherein a difference inconformation as compared to a reference single stranded polynucleotideindicates a mutation in the target ribonucleic acid. In otherembodiments, the invention provides methods of detecting a mutation (or,in some aspects, characterizing a sequence) in a target ribonucleic acidcomprising analyzing amplification products of any of the methodsdescribed herein for single stranded conformation, wherein a differencein conformation as compared to a reference single strandedpolynucleotide indicates a mutation in the target ribonucleic acid (or,in some aspects, characterizes the target sequence).

In another aspect, the invention provides methods of producing a nucleicacid immobilized to a substrate (which includes methods of producing amicroarray), comprising (a) amplifying a target RNA by any of themethods described herein; and (b) immobilizing the amplificationproducts on a substrate. The amplification products can be labeled orunlabeled. In other aspects, the invention provides methods of producinga microarray, comprising (a) amplifying a target RNA by an amplificationmethod described herein; and (b) immobilizing the amplification productson a substrate (which can be solid or semi-solid). In some embodiments,microarrays are produced by immobilizing amplification products onto asubstrate to make a microarray of amplification products. In otherembodiments, microarrays are produced by immobilizing amplificationproducts by any of the methods described herein onto a solid substrateto make a microarray of amplification products. The microarray cancomprise at least one amplification product immobilized on a solid orsemi-solid substrate fabricated from a material selected from the groupconsisting of paper, glass, ceramic, plastic, polypropylene, nylon,polyacrylamide, nitrocellulose, silicon an other metals, and opticalfiber. An amplification product can be immobilized on the solid orsemi-solid substrate in a two-dimensional configuration or athree-dimensional configuration comprising pins, rods, fibers, tapes,threads, beads, particles, microtiter wells, capillaries, and cylinders.

Any of the methods of the invention can be used to generatepolynucleotide (generally, RNA or DNA) products that are suitable forcharacterization of an RNA sequence of interest in a sample. In oneembodiment, the invention provides methods for characterizing (forexample, detecting and/or quantifying and/or determining presence orabsence of) an RNA sequence of interest comprising: (a) amplifying atarget RNA by any of the methods described herein; and (b) analyzing theamplification products. Step (b) of analyzing the amplification productscan be performed by any method known in the art or described herein, forexample by detecting and/or quantifying and/or determining present orabsence of amplification products that are hybridized to a probe. Theseamplification products may or may not be labeled. Any of the methods ofthe invention can be used to generate polynucleotide (generally, RNA orDNA) products that are labeled by incorporating labeled nucleotides intoappropriate step(s) of the methods. These labeled products areparticularly suitable for quantification and/or identification and/ordetermining presence or absence of by methods known in the art, whichinclude the use of arrays such as cDNA microarrays and oligonucleotidearrays. In one aspect, the invention provides a method of characterizingan RNA sequence of interest, comprising (a) amplifying a target RNA by amethod described herein to generate labeled products; and (b) analyzingthe labeled products. In some embodiments, the step of analyzing RNAproducts comprises determining amount of said products, whereby theamount of the RNA sequence of interest present in a sample isquantified. The polynucleotide products can be analyzed by, for example,contacting them with at least one probe. In some embodiments, the atleast one probe is provided as a microarray. The microarray can compriseat least one probe immobilized on a solid or semi-solid substratefabricated from a material selected from the group consisting of paper,glass, plastic, polypropylene, nylon, polyacrylamide, nitrocellulose,silicon, and optical fiber. A probe can be immobilized on the solid orsemi-solid substrate in a two-dimensional configuration or athree-dimensional configuration comprising pins, rods, fibers, tapes,threads, beads, particles, microtiter wells, capillaries, and cylinders.

In another aspect, the invention provides methods of determining geneexpression profile in a sample, the methods comprising: (a) amplifyingat least one RNA sequence of interest in the sample using any of themethods described herein; and (b) determining amount of amplificationproducts of each RNA sequence of interest, wherein each said amount isindicative of amount of each RNA sequence of interest in the sample,whereby the gene expression profile of the sample is determined.

In another aspect, the invention provides methods of preparing asubtractive hybridization probe, said methods comprising generatingmultiple single stranded polynucleotide, preferably DNA, copies of thecomplement of at least one RNA sequences of interest from a first RNApopulation using any of the methods described herein.

In another aspect, the invention provides methods of performingsubtractive hybridization, said methods comprising: (a) generatingmultiple copies of the complement of at least one RNA sequence ofinterest from a first RNA population using any of the methods describedherein; and (b) hybridizing the multiple copies to a second mRNApopulation, whereby a subpopulation of the second mRNA population formsa complex with a copy of the complement of at least one RNA sequence ofinterest. In embodiments in which DNA copies are generated, the methodsfurther comprise: (c) cleaving RNA in the complex of step (b) with anenzyme that cleaves RNA from an RNA/DNA hybrid; and (d) amplifying anunhybridized subpopulation of the second mRNA population (using anymethod, including the methods described herein), whereby multiple copiesof single stranded DNA complementary to the unhybridized subpopulationof the second mRNA population are generated.

In another aspect, the invention provides methods for differentialamplification, the methods comprising: (a) generating multiple nucleicacid (generally DNA) copies of the complement of at least one RNAsequence of interest from a first RNA population using any of themethods described herein; (b) hybridizing the multiple copies to asecond mRNA population, whereby a subpopulation of the second mRNApopulation forms a complex with a DNA copy; (c) cleaving RNA in thecomplex of step (b) with an enzyme that cleaves RNA from an RNA/DNAhybrid; and (d) amplifying an unhybridized subpopulation of the secondmRNA population using any method, including those described herein,whereby multiple copies of single stranded DNA complementary to theunhybridized subpopulation of the second mRNA population are generated.These methods encompass steps (b), (c) and (d) if the copies used in thesubtractive hybridization are generated using any of the methodsdescribed herein.

In another aspect, the invention provides methods for making a library,said method comprising preparing a subtractive hybridization probe asdescribed herein, or differential amplification as described herein. Anyof these applications can use any of the amplification methods(including various components and various embodiments of any of thecomponents) as described herein.

The invention also provides compositions, kits, complexes, reactionmixtures and systems comprising various components (and variouscombinations of the components) used in the amplification methodsdescribed herein. The compositions may be any component(s), reactionmixture and/or intermediate described herein, as well as any combinationthereof.

In some embodiments, the invention provides a composition comprising:(a) a first primer (which can be a random primer); (b) a second primer(which can be a random primer); and (c) a propromoter polynucleotide(which in some embodiments is a PTO). In some embodiments, thesecompositions may further comprise: (d) a third primer (which can be arandom primer). In some of these embodiments, the first primer comprisesa sequence that is not hybridizable to a target RNA. In some of theseembodiments, the second primer comprises a sequence that is nothybridizable to a first primer extension product. In some embodiments,the third primer comprises a sequence that is not hybridizable to an RNAtranscript. In some embodiments, the propromoter polynucleotide ((c),above) is capable of hybridizing to the complement of the 5′ portion ofthe first primer.

The invention also provides compositions comprising a propromoterpolynucleotide (such as a PTO) capable of hybridizing to a 3′ portion ofa second primer extension that is complement of a 5′ portion of a firstprimer used to create first primer extension product.

The invention also provides compositions comprising (a) a first primer;(b) a second primer (which can be a random primer); and (c) a compositeprimer, wherein the composite primer comprises a 5′ RNA portion and aDNA portion. In some embodiments, the invention provides a compositioncomprising: (a) a first primer (which can be a random primer)hybridizable to target RNA; (b) a second primer (which can be a randomprimer); and (c) a composite primer hybridizable to a second primerextension product. In some embodiments, the composition furthercomprises one or more of the following: DNA-dependent DNA polymerase,RNA-dependent DNA polymerase, and an agent (generally an enzyme) thatcleaves RNA from an RNA/DNA heteroduplex.

The invention also provides compositions comprising the amplificationproducts described herein. Accordingly, the invention provides apopulation of anti-sense RNA molecules which are copies of a targetsequence, which are produced by any of the methods described herein. Theinvention also provides a population of anti-sense polynucleotides(generally DNA) molecules, which are produced by any of the methodsdescribed herein.

In another aspect, the invention provides compositions comprising any ofthe complexes (which are generally considered as intermediates withrespect to the final amplification products) described herein (see alsothe figures for schematic depictions of examples of these variouscomplexes). For example, the invention provides compositions comprisinga complex of (a) a first primer extension product; and (b) a target RNAstrand. In yet another aspect, the invention provides compositionscomprising a complex of: (a) a first primer extension product; and (b) asecond primer extension product. In another example, the inventionprovides compositions comprising a complex of (a) a second primerextension product; and (b) a propromoter polynucleotide (which can be aPTO). In some embodiments, the propromoter polynucleotide hybridizes toa sequence in the second primer extension product comprising thecomplement of the 5′ portion of a first prime, wherein the first primeris extended to form the first primer extension product. In yet anotherexample, the invention provides compositions comprising a complex of (a)a third primer extension product; and (b) a propromoter polynucleotide(which can be a PTO). In yet another example, the invention providescompositions comprising a complex of (a) a second primer extensionproduct, generated by denaturation of a hybridized first and secondprimer extension product; and (b) a composite primer hybridizable to thesecond primer extension product.

In another aspect, the invention includes any one or more products(including intermediates) and compositions comprising the products(including intermediates) produced by any aspect of the methods of theinvention. The products include libraries and any other populationproduced, which are generally based on the nature of the primer(s) usedin the methods described herein.

In another aspect, the invention provides reaction mixtures (orcompositions comprising reaction mixtures) which contain variouscombinations of components described herein. For example, the inventionprovides reaction mixtures comprising (a) a target RNA; (b) a firstprimer; (c) a second primer (which can be a random primer); (d) an RNApolymerase; and (e) a DNA polymerase. The reaction mixture could alsofurther comprise an enzyme which cleaves RNA from an RNA/DNA hybrid,such as RNase H. A reaction mixture of the invention can also comprise apropromoter polynucleotide (which in some embodiments is a PTO).

In another aspect, the invention provides reaction mixtures comprising(a) a target RNA; (b) a first primer; (c) a second primer (which can bea random primer); (d) an RNA polymerase; (e) a DNA polymerase; and (f) acomposite primer. The reaction mixture could also further comprise anenzyme which cleaves RNA from an RNA/DNA hybrid, such as RNase H.

In another aspect, the invention provides kits for conducting themethods described herein. These kits, in suitable packaging andgenerally (but not necessarily) containing suitable instructions forperforming any of the methods of the invention described herein,including sequencing, detection of sequence alteration(s) (e.g.,genotyping or nucleic acid mutation detection); determining presence orabsence of a sequence of interest; gene expression profiling;subtractive hybridization; preparation of a subtractive hybridizationprobe; differential amplification; preparation of libraries (includingcDNA and differential expression libraries); preparation of animmobilized nucleic acid (which can be a nucleic acid immobilized on amicroarray), and characterizing (including detecting and/or quantifyingand/or determining presence or absence of) amplified nucleic acidproducts generated by the methods of the invention. The kits furthercomprise one or more components used in the methods of the invention.For example, the invention provides kits that comprise a first primerthat comprises a sequence the complement of which is hybridizable by apropromoter polynucleotide, and instructions for using the primer toamplify RNA. The invention also provides kits that further comprise asecond primer and/or a third primer, and optionally instructions forusing the primers to amplify RNA. The kits can contain furthercomponents, such as any of (a) a propromoter polynucleotide (such as aPTO); and (b) any of the enzymes described herein, such as an enzymewhich cleaves RNA from an RNA/DNA hybrid (for example, RNaseH), DNApolymerase (RNA-dependent or DNA-dependent) and RNA polymerase. Inanother example, a kit can comprise (a) a composite primer; and (b)instructions for using the composite primer to amplify target RNA usingthe methods of the invention provided herein. The kit can comprisefurther components, including any of the enzymes described herein, suchas an enzyme which cleaves RNA from an RNA/DNA hybrid (for example,RNaseH), and DNA polymerase (RNA-dependent or DNA-dependent). In anotherexample, a kit comprises a first primer that comprises a sequence thecomplement of which is hybridizable by a propromoter polynucleotide, andinstructions for using the primer to amplify RNA using any of themethods described herein. In another embodiment, the kit furthercomprises a second primer.

In another aspect, the invention provides systems for effecting theamplification methods described herein. For example, the inventionprovides systems for amplifying a target ribonucleic acid, comprising:(a) a first primer; (b) a second primer (which can be a random primer);(c) an RNA-dependent DNA polymerase; (d) a DNA-dependent DNA polymerase;(e) a propromoter polynucleotide (such as a PTO); and (f) an enzymewhich cleaves RNA from an RNA/DNA hybrid (such as RNaseH). The systemsmay also comprise: (g) a third primer (which can be a random primer). Asdescribed herein, systems of the invention generally comprise one ormore apparatuses appropriate for carrying out methods of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic representation of a linear RNA amplificationprocess. FIG. 1 shows amplification of a target RNA using apolynucleotide comprising a propromoter to produces multiple copies ofRNA transcripts complementary to the target RNA.

FIGS. 2A-B show further amplification of the RNA transcripts from theprocess of FIG. 1 to generate more RNA transcripts complementary to thetarget RNA.

FIGS. 3A-B show a diagrammatic representation of a linear RNAamplification process using a third primer that is a composite primer togenerate single stranded DNA strands complementary to the target RNA.

MODES FOR CARRYING OUT THE INVENTION

The invention provides methods, compositions and kits for amplifyingpolynucleotide sequences, specifically ribonucleic acid (RNA) sequences.The methods provide for amplification of a single RNA species or pool ofRNA species. The methods can achieve exponential amplification, whichwould be particularly useful for amplification of very low amounts ofRNA sequences in a biological sample. The methods are suitable for, forexample, generation of libraries, including cDNA libraries. Theygenerate single stranded RNA or, in some embodiments, single strandedDNA products, which are readily suitable for multiplex analysis bymicroarray technologies, as well as electrophoresis-based technologiessuch as differential display. The methods are amenable to automation anddo not require thermal cycling. The methods generally comprisehybridizing a polynucleotide comprising a propromoter sequence to aprimer extension product to generate an intermediate product capable ofdriving transcription, whereby RNA transcripts comprising sequencescomplementary to an RNA sequence of interest are produced. In anotheraspect, the methods comprise isothermal linear amplification of DNAcopies complementary to the RNA sequence of interest using adenaturation step, a composite primer and strand displacement. See Kurn,U.S. Pat. No. 6,251,639 B1.

The methods of the invention are directed to the amplification of one ormore species of RNA, such as a pool of RNA sequences, and is mostparticularly suitable for the amplification of all RNA (such as mRNA)sequences in a preparation of total RNA from a biological sample. Thus,one of the major advantages of the methods of the invention is theability to amplify an entire pool of sequences, which is essential forthe ability to analyze the gene expression profile in cells, such as thecells in a biological of interest sample. The methods of the inventionhave the potential of amplifying a multiplicity, more preferably a largemultiplicity, and most preferably all RNA (such as mRNA) sequences in asample.

Insofar as many mRNAs have a unique polyA 3′-end, the amplificationinitiated from the 3′-end sequence of mRNAs is most common forpreparation of cDNA libraries and subsequent sequence analysis fordetermination of gene expression profiling or other applications. Themethods of the invention are similarly suited for preparation oflibraries of amplified 3′-portions of mRNAs. The sequence of the firstprimer used in the methods of invention can be designed to becomplementary to a multiplicity, or all, of the mRNA species in thesample by using random sequences, according to methods known in the art.

Various methods for mRNA amplification have been described. U.S. Pat.Nos. 744,308; 6,143,495; EP 0971039A2; EP 0878553A2. Most of thesemethods are transcription based, wherein a promoter for RNA polymeraseis incorporated into a double stranded cDNA by a primer comprising apropromoter sequence at the 5′-end which hybridizes to target RNA. Theseprimers can non-specifically bind to template RNA. Insofar as a DNApolymerase has a high affinity for primer hybridized to a templatenucleic acid with a free 3′ end, i.e. a substrate for primer extensionby the polymerase, it is highly probable that a primer comprising apropromoter sequence at the 5′ end may non-specifically incorporate thepromoter sequence into an amplification product. This results inuncontrolled production of transcription products. The appending of adouble stranded promoter by a propromoter polynucleotide, as describedherein, provides for increased specificity and control of thetranscription-based generation of amplification product.

In one aspect, the invention works as follows: generation of multiplecopies of the complementary sequence of an RNA sequence of interest isachieved by using a first primer (which can be a specific or randomprimer) that comprises a sequence (generally, in its 5′ portion) thecomplement of which is hybridizable by a polynucleotide comprising apropromoter. In some embodiments, the sequence the complement of whichis hybridizable by a polynucleotide comprising a propromoter is asequence that is hybridizable to a target RNA when the primer ishybridized to the target RNA. In other embodiments, the sequence thecomplement of which is hybridizable by a polynucleotide comprising apropromoter is a sequence that is not hybridizable to a target RNA whenthe primer is hybridized to the target RNA (thus forming a tail when theprimer is hybridized to a target). The extension of the first primeralong a target RNA by an RNA-dependent DNA polymerase results in thegeneration of an intermediate polynucleotide (first primer extensionproduct) that has at least one defined end (the first primer end). Aftercleavage of the template RNA from a complex comprising the target RNAand first primer extension product, a second primer (which can be aspecific or random primer) is then hybridized to the first DNA strand(first primer extension product) and extended to form a complex of firstand second primer extension products that at one end comprises asequence to which a polynucleotide comprising a propromoter ishybridizable. The second primer is any sequence that is hybridized tothe first DNA strand such that it is capable of being extended by a DNApolymerase along a first DNA strand to generate a second DNA strand.Thus, in some embodiments, the second primer is an oligonucleotide (thatis separately provided). In other embodiments, it is or comprises asequence of the first DNA strand (generally at the 3′ end) that ishybridized to a sequence in the first DNA strand (for example, a hairpinor self-annealed structure). Following denaturation of the complex, apolynucleotide comprising a propromoter is hybridized to the secondprimer extension product and the 3′ end of the primer extension productis extended along the propromoter oligonucleotide (if there is anyoverhang) to generate a double stranded promoter region. RNAtranscription driven by this promoter results in generation of multiplecopies of RNA transcripts comprising the complementary sequence of theRNA sequence of interest. In some embodiments involving cyclicalamplification (also referred to herein as “exponential” amplification),these RNA transcripts, and optionally RNA transcripts generated insubsequent steps, are hybridized with a third primer (which may or maynot be the same as the second primer, and which may be a specific orrandom primer). The third primer is extended to form a complexcomprising the RNA transcript and a third primer extension product(which constitutes a DNA/RNA heteroduplex). Cleavage of the RNAtranscript results in a single stranded third primer extension productto which a propromoter polynucleotide hybridizes. If necessary (i.e., ifthere is an overhang), the third primer extension product is extendedalong the propromoter polynucleotide to generate a double strandedpromoter region. RNA transcription driven by this promoter results ingeneration of multiple copies of RNA transcripts comprising thecomplementary sequences of the RNA sequence of interest. Hybridizationof third primers to these RNA transcripts initiates a cyclical processleading to further amplification.

Accordingly, the invention provides methods of producing at least onecopy of the complementary sequence of an RNA sequence of interest, saidmethod comprising combining and reacting the following: (a) a target RNAcomprising an RNA sequence of interest; (b) a first primer thathybridizes to the target RNA; (c) a second primer that is hybridizableto an extension product of the first primer; (d) an RNA-dependent DNApolymerase; (e) a DNA-dependent DNA polymerase; (f) an enzyme thatcleaves RNA from an RNA/DNA hybrid; (g) a propromoter polynucleotidecomprising a propromoter and a region which hybridizes to an extensionproduct of the second primer; (h) deoxyribonucleoside triphosphates orsuitable analogs; (i) ribonucleoside triphosphates and suitable analogs;and (j) an RNA polymerase. In embodiments involving cyclicalamplification (interchangeably termed “exponential” amplification,herein), the following are also included in the amplification reaction(either at the same time as the components listed above or addedseparately): (k) optionally a third primer (which may or may not be thesame as the second primer) that hybridizes to an RNA transcriptcomprising sequences complementary to the sequence of the target RNA;and (l) optionally a second polynucleotide comprising a propromoter anda region which hybridizes to a single stranded third primer extensionproduct (this polynucleotide may or may not be the same as thepolynucleotide described in (g) above).

As is evident to one skilled in the art, by this disclosure, thereactions described may be simultaneous or sequential, as such, theinvention includes these various embodiments and combinations.

In some embodiments, the invention provides methods of producing atleast one copy of the complementary sequence of an RNA sequence ofinterest, said method comprising combining and reacting the following:(a) a single stranded second primer extension product resulting fromdenaturation of a complex of first and second primer extension productsas described herein; (b) a propromoter polynucleotide comprising apropromoter and a region which hybridizes to a second primer extensionproduct; (c) ribonucleoside triphosphates and suitable analogs; and (d)an RNA polymerase. In embodiments involving cyclical amplification(“exponential” amplification), the following may also included in theamplification reaction (either at the same time as the components listedabove or added separately): (e) optionally a third primer (which may ormay not be the same as the second primer) that hybridizes to an RNAtranscript comprising sequences complementary to the sequence of thetarget RNA; and (f) optionally a second polynucleotide comprising apropromoter and a region which hybridizes to a single stranded thirdprimer extension product (this polynucleotide may or may not be the sameas the polynucleotide described in (b) above). In some embodimentsinvolving cyclical amplification, said method comprises combining andreacting (under suitable conditions and reagent such that multiplecopies of a polynucleotide sequence complementary to an RNA sequence ofinterest are produced) the following: (a) an RNA transcript generatedfrom the complex of the first propromoter polynucleotide and secondprimer extension product; (b) a third primer (which may or may not bethe same as the second primer) that hybridizes to the RNA transcript;(c) a second polynucleotide comprising a propromoter and a region whichhybridizes to a single stranded third primer extension product (thispolynucleotide may or may not be the same as the propromoterpolynucleotide in the complex from which the RNA transcript in step (a)is generated).

In another aspect, the invention provides a method of generatingmultiple copies of a polynucleotide sequence complementary to an RNAsequence of interest as follows: generation of multiple copies of thecomplementary sequence of an RNA sequence of interest is achieved byusing a first primer (which can be a specific or random primer) thatcomprises a sequence in its 5′ portion that is not hybridizable totarget RNA (thus forming a tail when the primer is hybridized to atarget under conditions when the first primer hybridizes to templateRNA). The extension of the first primer along a target RNA by anRNA-dependent DNA polymerase results in the generation of anintermediate polynucleotide (first strand cDNA) that has at least onedefined end comprising the complement of the first primer sequence.After cleavage of the template RNA from a complex comprising the targetRNA and first strand cDNA, a second primer (which can be a specific orrandom primer) is then hybridized to the first strand cDNA) and extendedto form a complex of first and second strand cDNAs that at (at least)one end has a defined end comprising the first primer sequence and thecomplement of the first primer sequence. The second primer is anysequence that is hybridized to the first DNA strand such that it iscapable of being extended by a DNA polymerase along a first DNA strandto generate a second DNA strand. Thus, in some embodiments, the secondprimer is an oligonucleotide (that is separately provided). In otherembodiments, it comprises a sequence of the first DNA strand (generallyat the 3′ end) that is hybridized to a sequence in the first DNA strand(for example, a hairpin or self-annealed structure). In otherembodiment, the second primer comprises one or more fragments of thetarget RNA sequence that remains hybridized to the first primerextension product (after cleavage of the initial complex comprising thetarget RNA and first primer extension product). The complex of first andsecond strand cDNAs (wherein the second strand cDNA comprises a 3′ endportion that is the complement of the first primer sequence) is thendenatured to form single stranded first strand cDNA and second strandcDNA.

The single stranded second strand cDNA is the substrate for isothermallinear amplification using a composite primer and strand displacement asfollows: a composite primer comprising a 5′-RNA portion and a DNAportion hybridizes to the 3′-portion of the second strand cDNA,generally to the 3′-portion of the second strand cDNA, and is extendedalong the second strand cDNA by a DNA polymerase to form a doublestranded complex comprising an RNA/DNA hybrid portion at one end of thecomplex. An enzyme which cleaves RNA from an RNA/DNA hybrid (such asRNase H) cleaves RNA sequence from the hybrid, leaving a sequence on thesecond strand cDNA available for binding by another composite primer.Another first (composite) strand cDNA is produced by DNA polymerase,which displaces the previously bound cleaved first strand cDNA,resulting in displaced cleaved first strand cDNA. The displaced cleavedfirst strand cDNA product comprises single stranded DNA complementary tothe RNA sequence of interest. Kurn, U.S. Pat. No. 6,251,639 B1.

Any of the methods of the invention can be used to generatepolynucleotide (generally, RNA or DNA) products that are labeled byincorporating labeled nucleotides into appropriate steps of the methods.These labeled products are particularly suitable for quantificationand/or identification by methods known in the art, which include the useof arrays such as cDNA microarrays and oligonucleotide arrays.

In some embodiments, the invention provides methods of sequencing RNAsequences. For sequencing methods based on methods described herein, theappropriate rNTPs, or analogs thereof, which may be labeled orunlabeled, are used. Accordingly, the invention provides methods ofsequencing a target RNA comprising a sequence of interest based on themethods described above, wherein rNTPs and rNTP analogs which are primerelongation terminators, which may be labeled or unlabeled, are used, andthe amplification product is analyzed for sequence information, asdescribed below. For sequencing methods based on methods describedherein wherein the amplified product is DNA, the appropriate dNTPs, oranalogs thereof, which may be labeled or unlabeled, are used.

In other embodiments, the invention provides methods of detectingnucleic acid sequence mutations. In one embodiment, the amplificationproducts are used to detect and/or identify single strand conformationpolymorphisms in a target RNA.

The invention provides methods to characterize (for example, detectand/or quantify and/or determine presence or absence of) an RNA sequenceof interest by generating polynucleotide (generally RNA or DNA) productsusing amplification methods of the invention, and analyzing the productsby detection/quantification methods such as those based on arraytechnologies or solution phase technologies. Generally, but notnecessarily, these amplified products are labeled.

In another aspect, the invention provides a method of characterizing anRNA sequence of interest, comprising (a) amplifying a target RNA by amethod described herein to generate labeled products; and (b) analyzingthe labeled polynucleotide (generally, RNA or DNA) products. In someembodiments, the step of analyzing products comprises determining amountof said products, whereby the amount of the RNA sequence of interestpresent in a sample is quantified. The polynucleotide (generally, DNA orRNA) products can be analyzed by, for example, contacting them with atleast one probe. In some embodiments, the at least one probe is providedas a microarray. The microarray can comprise at least one probeimmobilized on a solid or semi-solid substrate fabricated from amaterial selected from the group consisting of paper, glass, ceramics,plastic, polypropylene, polystyrene, nylon, polyacrylamide,nitrocellulose, silicon, other metals, and optical fiber. A probe can beimmobilized on the solid or semi-solid substrate in a two-dimensionalconfiguration or a three-dimensional configuration comprising pins,rods, fibers, tapes, threads, beads, particles, microtiter wells,capillaries, and cylinders.

In another aspect, the invention provides methods of determining geneexpression profile in a sample, the methods comprising: (a) amplifyingat least one RNA sequence of interest in the sample using any of themethods described herein; and (b) determining amount of amplificationproducts of each RNA sequence of interest, wherein each said amount isindicative of amount of each RNA sequence of interest in the sample,whereby the gene expression profile of the sample is determined.

In another embodiment, the invention provides methods of generatinglibraries (including cDNA libraries and subtractive hybridizationlibraries), said methods comprising: amplifying at least one RNAsequences of interest using any of the methods described herein togenerate single stranded nucleic acid product, and methods of generatingand using subtractive hybridization probes, methods for subtractivehybridization, methods for differential amplification, and methods ofgenerating subtractive hybridization libraries.

Various methods for the detection and quantification of gene expressionlevels have been developed in recent years. For example, microarrays, inwhich either defined cDNAs or oligonucleotides are immobilized atdiscrete locations on, for example, solid or semi-solid substrates, oron defined particles, enables the detection and/or quantification of theexpression of a multitude of genes in a given specimen.

Using these previously known methods to detect and/or quantify multiplemRNA species in a sample, which in turn is a measure of gene expressionprofiling, generally requires direct labeling of cDNA, which requires alarge amount of input total RNA, in part because mRNA represents only asmall subset of the total RNA pool. Thus, when using total RNApreparations from a given cell or tissue sample, the analysis of geneexpression in the sample using methods such as arrays requires asubstantial amount of input RNA, which generally ranges from 50 to 200μg. Similarly, 2 to 5 μg of mRNA purified from a total RNA preparationwould generally be required for a single analysis of gene expressionprofiling using array technologies. This is a clear limitation ofmethods such as those based on array technology, insofar as the numberof cells, or size of tissue specimen required is very large, and thesecells or tissue specimens are often scarcely available for testing orare too precious. This limitation is especially severe in the study ofclinical specimens, where the cells to be studied are rare and/ordifficult to cultivate in vitro, and in high throughput screening oflibraries of effector molecules.

General Techniques

The practice of the invention will employ, unless otherwise indicated,conventional techniques of molecular biology (including recombinanttechniques), microbiology, cell biology, biochemistry, and immunology,which are within the skill of the art. Such techniques are explainedfully in the literature, such as, “Molecular Cloning: A LaboratoryManual”, second edition (Sambrook et al., 1989); “OligonucleotideSynthesis” (M. J. Gait, ed., 1984); “Animal Cell Culture” (R. I.Freshney, ed., 1987); “Methods in Enzymology” (Academic Press, Inc.);“Current Protocols in Molecular Biology” (F. M. Ausubel et al., eds.,1987, and periodic updates); “PCR: The Polymerase Chain Reaction”,(Mullis et al., eds., 1994).

Primers, oligonucleotides and polynucleotides employed in the inventioncan be generated using standard techniques known in the art.

Definitions

A “target sequence,” “target nucleic acid,” or “target RNA,” as usedherein, is a polynucleotide comprising a sequence of interest, for whichamplification is desired. The target sequence may be known or not known,in terms of its actual sequence. In some instances, the terms “target”and “template”, and variations thereof, are used interchangeably.

“Amplification,” as used herein, generally refers to the process ofproducing multiple copies of a desired sequence. “Multiple copies” meanat least 2 copies. A “copy” does not necessarily mean perfect sequencecomplementarity or identity to the template sequence. For example,copies can include nucleotide analogs such as deoxyinosine, intentionalsequence alterations (such as sequence alterations introduced through aprimer comprising a sequence that is hybridizable, but notcomplementary, to the template, or a non-target sequence introducedthrough a primer), and/or sequence errors that occur duringamplification. “Amplifying” a sequence may generally refer to makingcopies of a sequence or its complement, with the understanding that, asstated above, copying does not necessarily mean perfect sequencecomplementarity or identity with respect to the template sequence.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase. A polynucleotidemay comprise modified nucleotides, such as methylated nucleotides andtheir analogs. If present, modification to the nucleotide structure maybe imparted before or after assembly of the polymer. The sequence ofnucleotides may be interrupted by non-nucleotide components. Apolynucleotide may be further modified after polymerization, such as byconjugation with a labeling component. Other types of modificationsinclude, for example, “caps”, substitution of one or more of thenaturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, cabamates, etc.)and with charged linkages (e.g., phosphorothioates, phosphorodithioates,etc.), those containing pendant moieties, such as, for example, proteins(e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine,etc.), those with intercalators (e.g., acridine, psoralen, etc.), thosecontaining chelators (e.g., metals, radioactive metals, boron, oxidativemetals, etc.), those containing alkylators, those with modified linkages(e.g., alpha anomeric nucleic acids, etc.), as well as unmodified formsof the polynucleotide(s). Further, any of the hydroxyl groups ordinarilypresent in the sugars may be replaced, for example, by phosphonategroups, phosphate groups, protected by standard protecting groups, oractivated to prepare additional linkages to additional nucleotides, ormay be conjugated to solid supports. The 5′ and 3′ terminal OH can bephosphorylated or substituted with amines or organic capping groupsmoieties of from 1 to 20 carbon atoms. Other hydroxyls may also bederivatized to standard protecting groups. Polynucleotides can alsocontain analogous forms of ribose or deoxyribose sugars that aregenerally known in the art, including, for example, 2′-O-methyl-,2′-O-allyl, 2′-fluoro- or 2′-azido-ribose, carbocyclic sugar analogs,α-anomeric sugars, epimeric sugars such as arabinose, xyloses orlyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclicanalogs and abasic nucleoside analogs such as methyl riboside. One ormore phosphodiester linkages may be replaced by alternative linkinggroups. These alternative linking groups include, but are not limitedto, embodiments wherein phosphate is replaced by P(O)S(“thioate”), P(S)S(“dithioate”), “(O)NR₂ (“amidate”), P(O)R, P(O)OR′, CO or CH₂(“formacetal”), in which each R or R′ is independently H or substitutedor unsubstituted alkyl (1-20 C) optionally containing an ether (—O—)linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not alllinkages in a polynucleotide need be identical. The precedingdescription applies to all polynucleotides referred to herein, includingRNA and DNA.

A “labeled dNTP,” or “labeled rNTP,” as used herein, refers,respectively, to a dNTP or rNTP, or analogs thereof, that is directly orindirectly attached with a label. For example, a “labeled” dNTP or rNTP,may be directly labeled with, for example, a dye and/or a detectablemoiety, such as a member of a specific binding pair (such asbiotin-avidin). A “labeled” dNTP or rNTP, may also be indirectly labeledby its attachment to, for example, a moiety to which a label is/can beattached. A dNTP or rNTP, may comprise a moiety (for example, an aminegroup) to which a label may be attached following incorporation of thedNTP or rNTP into an extension product. Useful labels in the presentinvention include fluorescent dyes (e.g., fluorescein isothiocyanate,Texas red, rhodamine, green fluorescent protein and the like),radioisotopes (e.g., ³H, ³⁵S, ³²P, ³³P, ¹²⁵I, or ¹⁴C), enzymes (e.g.,LacZ, horseradish peroxidase, alkaline phosphatase), digoxigenin, andcolorimetric labels such as colloidal gold or colored glass or plastic(e.g., polystyrene, polypropylene, latex, etc.) beads. Variousanti-ligands and ligands can be used (as labels themselves or as a meansfor attaching a label). In the case of a ligand that has a naturalanti-ligand, such as biotin, thyroxine and cortisol, the ligand can beused in conjunction with labeled anti-ligands.

The “type” of dNTP or rNTP, as used herein, refers to the particularbase of a nucleotide, namely adenine, cytosine, thymine, uridine, orguanine.

“Oligonucleotide,” as used herein, generally refers to short, generallysingle stranded, generally synthetic polynucleotides that are generally,but not necessarily, less than about 200 nucleotides in length.Oligonucleotides in the invention include the first, second, and thirdprimers, and propromoter polynucleotide (such as the PTO). The terms“oligonucleotide” and “polynucleotide” are not mutually exclusive. Thedescription above for polynucleotides is equally and fully applicable tooligonucleotides.

A “primer” is generally a nucleotide sequence (i.e. a polynucleotide),generally with a free 3′-OH group, that hybridizes with a templatesequence (such as a target RNA, or a primer extension product) and iscapable of promoting polymerization of a polynucleotide complementary tothe template. A “primer” can be, for example, an oligonucleotide. It canalso be, for example, a sequence of the template (such as a primerextension product or a fragment of the template created following RNasecleavage of a template-DNA complex) that is hybridized to a sequence inthe template itself (for example, as a hairpin loop), and that iscapable of promoting nucleotide polymerization. Thus, a primer can be anexogenous (e.g., added) primer or an endogenous (e.g., templatefragment) primer.

A “random primer,” as used herein, is a primer that comprises a sequencethat is designed not necessarily based on a particular or specificsequence in a sample, but rather is based on a statistical expectation(or an empirical observation) that the sequence of the random primer ishybridizable (under a given set of conditions) to one or more sequencesin the sample. The sequence of a random primer (or its complement) mayor may not be naturally-occurring, or may or may not be present in apool of sequences in a sample of interest. The amplification of aplurality of RNA species in a single reaction mixture would generally,but not necessarily, employ a multiplicity, preferably a largemultiplicity, of random primers. As is well understood in the art, a“random primer” can also refer to a primer that is a member of apopulation of primers (a plurality of random primers) which collectivelyare designed to hybridize to a desired and/or a significant number oftarget sequences. A random primer may hybridize at a plurality of siteson a nucleic acid sequence. The use of random primers provides a methodfor generating primer extension products complementary to a targetpolynucleotide which does not require prior knowledge of the exactsequence of the target.

“Protopromoter sequence,” and “propromoter sequence,” as used herein,refer to a single-stranded DNA sequence region which, in double-strandedform is capable of mediating RNA transcription. In some contexts,“protopromoter sequence,” “protopromoter,” “propromoter sequence,”“propromoter,” “promoter sequence,” and “promoter” are usedinterchangeably.

A “propromoter polynucleotide,” as used herein, refers to apolynucleotide comprising a propromoter sequence. Example of apropromoter polynucleotide is a propromoter template oligonucleotide(PTO).

“Propromoter template oligonucleotide (PTO)” and “promoter templateoligionucleotide (PTO)” as used herein, refer to an oligonucleotide thatcomprises a propromoter sequence and a portion, generally a 3′ portion,that is hybridizable (under a given set of conditions) to the 3′ regionof a primer extension product. The propromoter sequence and thehybridizable portion may be the same, distinct or overlappingnucleotides of an oligonucleotide.

To “inhibit” is to decrease or reduce an activity, function, and/oramount as compared to a reference.

A “complex” is an assembly of components. A complex may or may not bestable and may be directly or indirectly detected. For example, as isdescribed herein, given certain components of a reaction, and the typeof product(s) of the reaction, existence of a complex can be inferred.For purposes of this invention, a complex is generally an intermediatewith respect to the final amplification product(s). An example of acomplex is a nucleic acid duplex comprising a first primer extensionproduct and a second primer extension product.

“Denaturing,” or “denaturation of” a complex comprising twopolynucleotides (such as a first primer extension product and a secondprimer extension product) refers to dissociation of two hybridizedpolynucleotide sequences in the complex. The dissociation may involve aportion or the whole of each polynucleotide. Thus, denaturing ordenaturation of a complex comprising two polynucleotides can result incomplete dissociation (thus generating two single strandedpolynucleotides), or partial dissociation (thus generating a mixture ofsingle stranded and hybridized portions in a previously double strandedregion of the complex).

A “portion” or “region,” used interchangeably herein, of apolynucleotide or oligonucleotide is a contiguous sequence of 2 or morebases. In other embodiments, a region or portion is at least about anyof 3, 5, 10, 15, 20, 25 contiguous nucleotides.

A region, portion, or sequence which is “adjacent” to another sequencedirectly abuts that region, portion, or sequence.

A “reaction mixture” is an assemblage of components, which, undersuitable conditions, react to form a complex (which may be anintermediate) and/or a product(s).

“A”, “an” and “the”, and the like, unless otherwise indicated includeplural forms.

“Comprising” means including.

Conditions that “allow” an event to occur or conditions that are“suitable” for an event to occur, such as hybridization, strandextension, and the like, or “suitable” conditions are conditions that donot prevent such events from occurring. Thus, these conditions permit,enhance, facilitate, and/or are conducive to the event. Such conditions,known in the art and described herein, depend upon, for example, thenature of the nucleotide sequence, temperature, and buffer conditions.These conditions also depend on what event is desired, such ashybridization, cleavage, strand extension or transcription.

Sequence “mutation,” as used herein, refers to any sequence alterationin a sequence of interest in comparison to a reference sequence. Areference sequence can be a wild type sequence or a sequence to whichone wishes to compare a sequence of interest. A sequence mutationincludes single nucleotide changes, or alterations of more than onenucleotide in a sequence, due to mechanisms such as substitution,deletion or insertion. Single nucleotide polymorphism (SNP) is also asequence mutation as used herein.

“Single stranded conformation polymorphism,” and “SSCP,” as used herein,generally refer to the specific conformation of a single strandednucleic acid as is affected by its specific nucleic acid sequence.Alteration of the sequence of the single stranded polynucleotide, suchas single nucleotide substitution, deletions or insertions, result inchange, or polymorphism, of the conformation of the single strandedpolynucleotide. The conformation of the polynucleotide is generallydetectable, identifiable and/or distinguishable using methods known inthe art, such as electrophoretic mobility as measured by gelelectrophoresis, capillary electrophoresis, and/or susceptibility toendonuclease digestion.

“Microarray” and “array,” as used interchangeably herein, comprise asurface with an array, preferably ordered array, of putative binding(e.g., by hybridization) sites for a biochemical sample (target) whichoften has undetermined characteristics. In a preferred embodiment, amicroarray refers to an assembly of distinct polynucleotide oroligonucleotide probes immobilized at defined positions on a substrate.Arrays are formed on substrates fabricated with materials such as paper,glass, ceramic, plastic (e.g., polypropylene, nylon, polystyrene),polyacrylamide, nitrocellulose, silicon or other metals, optical fiberor any other suitable solid or semisolid support, and configured in aplanar (e.g., glass plates, silicon chips) or three-dimensional (e.g.,pins, fibers, beads, particles, microtiter wells, capillaries)configuration. Probes forming the arrays may be attached to thesubstrate by any number of ways including (i) in situ synthesis (e.g.,high-density oligonucleotide arrays) using photolithographic techniques(see, Fodor et al., Science (1991), 251:767-773; Pease et al., Proc.Natl. Acad. Sci. U.S.A. (1994), 91:5022-5026; Lockhart et al., NatureBiotechnology (1996), 14:1675; U.S. Pat. Nos. 5,578,832; 5,556,752; and5,510,270); (ii) spotting/printing at medium to low-density (e.g., cDNAprobes) on glass, nylon or nitrocellulose (Schena et al, Science (1995),270:467-470, DeRisi et al, Nature Genetics (1996), 14:457-460; Shalon etal., Genome Res. (1996), 6:639-645; and Schena et al., Proc. Natl. Acad.Sci. U.S.A. (1995), 93:10539-11286); (iii) by masking (Maskos andSouthern, Nuc. Acids. Res. (1992), 20:1679-1684) and (iv) bydot-blotting on a nylon or nitrocellulose hybridization membrane (see,e.g., Sambrook et al., Eds., 1989, Molecular Cloning: A LaboratoryManual, 2nd ed., Vol. 1-3, Cold Spring Harbor Laboratory (Cold SpringHarbor, N.Y.)). Probes may also be noncovalently immobilized on thesubstrate by hybridization to anchors, by means of magnetic beads, or ina fluid phase such as in microtiter wells or capillaries. The probemolecules are generally nucleic acids such as DNA, RNA, PNA, and cDNAbut may also include proteins, polypeptides, oligosaccharides, cells,tissues and any permutations thereof which can specifically bind thetarget molecules.

The term “3′” generally refers to a region or position in apolynucleotide or oligonucleotide 3′ (downstream) from another region orposition in the same polynucleotide or oligonucleotide.

The term “5′” generally refers to a region or position in apolynucleotide or oligonucleotide 5′ (upstream) from another region orposition in the same polynucleotide or oligonucleotide.

The term “3′-DNA portion,” “3′-DNA region,” “3′-RNA portion,” and“3′-RNA region,” refer to the portion or region of a polynucleotide oroligonucleotide located towards the 3′ end of the polynucleotide oroligonucleotide, and may or may not include the 3′ most nucleotide(s) ormoieties attached to the 3′ most nucleotide of the same polynucleotideor oligonucleotide. The 3′ most nucleotide(s) can be preferably fromabout 1 to about 20, more preferably from about 3 to about 18, even morepreferably from about 5 to about 15 nucleotides.

The term “5′-DNA portion,” “5′-DNA region,” “5′-RNA portion,” and“5′-RNA region,” refer to the portion or region of a polynucleotide oroligonucleotide located towards the 5′ end of the polynucleotide oroligonucleotide, and may or may not include the 5′ most nucleotide(s) ormoieties attached to the 5′ most nucleotide of the same polynucleotideor oligonucleotide. The 5′ most nucleotide(s) can be preferably fromabout 1 to about 20, more preferably from about 3 to about 18, even morepreferably from about 5 to about 15 nucleotides.

As is well understood by those skilled in the art, a “tail” sequence ofa primer is a sequence not hybridizable to the target sequence underconditions in which other region(s) or portion(s) of the primerhybridizes to the target.

Amplifcation Methods of the Invention

The following are examples of the amplification methods of theinvention. It is understood that various other embodiments may bepracticed, given the general description provided above.

Methods of generating multiple copies of (amplifying) an RNA sequencecomplementary to an RNA sequence of interest are provided. In someaspect, the amplification methods of the invention include atranscription step. In a first embodiment of these methods, linearnucleic acid amplification is achieved based on hybridizing apropromoter polynucleotide to a primer extension product to generate anintermediate product capable of driving transcription, whereby RNAtranscripts comprising sequences complementary to an RNA sequence ofinterest are produced. In a second embodiment of these methods,exponential amplification is achieved by subjecting amplified RNAproducts generated in the process of the first embodiment of this methodand in subsequent amplification to cyclical amplification.

In embodiments of the amplification methods of the invention whichinclude a transcription step generally provide as follows: if a primerextension product that is to be transcribed comprises a propromotersequence, a double stranded promoter region is generally generated byhybridizing a polynucleotide comprising a propromoter (also referred toherein as “propromoter polynucleotide”) to the primer extension product.If a primer extension product does not comprise a desired propromotersequence, the transcription step is generally dependent on theincorporation of an RNA polymerase propromoter sequence, by use of apropromoter polynucleotide such as a promoter sequence oligonucleotide(PTO). A propromoter polynucleotide such as the PTO can serve as atemplate for extension of a single stranded primer extension product andformation of a partial duplex comprising a double stranded promoter atone end. The ability to hybridize a single stranded primer extensionproduct to the propromoter polynucleotide (such as a PTO) is generallyachieved by creating a primer extension product with a defined 3′ endsequence, which is complementary to the 3′ end sequence of thepropromoter polynucleotide.

In another aspect, the invention provides a method for generatingmultiple copies (amplifying) of a polynucleotide (DNA) sequencecomplementary to an RNA sequence of interest using a first primer and acomposite primer.

One aspect of the methods of the invention includes the design ofprimers which are able to hybridize to RNA sequences, such as aplurality of RNA sequences, for initiation of primer extension andproduction of amplification substrates and products.

Methods of Amplifying an RNA Sequence of Interest

Nucleic Acid Sequence Amplification Using a First Primer and aPropromoter Polynucleotide, and a Propromoter Polynucleotide

The invention provides methods of generating multiple copies of thecomplementary sequence of an RNA sequence of interest by using a primerthat comprises a sequence that when introduced into the amplificationsteps result in generation of an intermediate polynucleotide to which apropromoter polynucleotide can hybridize. The primer is designed tocomprise a sequence the complement of which is hybridizable by apropromoter polynucleotide. Generally, this sequence is in the 5′portion of the primer. The sequence can be a sequence (the complement ofwhich is hybridizable by the propromoter polynucleotide used) that ishybridizable to a target RNA, or a sequence (the complement of which ishybridizable by the propromoter polynucleotide used) that is nothybridizable to a target RNA. In some embodiments, linear amplificationis achieved. In other embodiments wherein amplified RNA products aresubjected to further rounds of amplification, cyclical, and thusexponential, amplification is achieved.

A schematic exemplary depiction of one embodiment of the linear methodsof the invention is provided in FIG. 1. A schematic depiction of oneembodiment of the exponential amplification methods of the invention isprovided in FIG. 2. An embodiment of the linear amplification method ofthe invention illustrated in FIG. 1 employs two oligonucleotides whichare combined with the sample as shown in the figure: a) a first primer(labeled “4”), which can be composed of two portions, a 3′ portion(labeled “A”), and a 5′ portion (labeled “B”); and b) a second primer(labeled 5”). The exponential method of the invention as illustrated inFIGS. 2A-B employs three oligonucleotides which are combined with thesample as shown in the figure: a) a first primer (labeled “4”) which canbe composed of two portions, a 3′ portion (labeled “A”); and a 5′portion (labeled “B”); b) a second primer (labeled “5”); and c) apropromoter polynucleotide, such as a promoter template oligonucleotide(PTO), (labeled “6”).

The 3′ portion of the first primer illustrated in FIGS. 1 and 2A can bedesigned in any of a number of ways (in terms of sequence), depending onwhich type, class, population, and/or species of RNA is desired to beamplified. In some embodiments, the 3′ portion of the first primer, asillustrated in FIGS. 1 and 2A, comprises a sequence complementary to thepoly-A tail of mRNA, and may further comprise additional randomsequences (generally not complementary to a poly-A sequence) at the 3′end of the 3′ portion. In other embodiments, the 3′ portion of the firstprimer is a random primer comprising sequences which are hybridizable toa multiplicity of RNA species (which may range from 2 or more to manyhundred or thousands or more). Random primers are known in the art, forexample, they have been used extensively in the preparation of cDNAlibraries using PCR-based procedures. As is well understood in the art,a “random primer” can refer to a primer that is a member of a populationof primers (a plurality of random primers) which collectively aredesigned to hybridize to a desired and/or a significant number of targetsequences. A random primer may hybridize at a plurality of sites on anucleic acid sequence.

In other embodiments, the 3′ portion of the first primer can comprise asequence complementary or hybridizable to a specific RNA or family ofRNAs (or portions thereof).

In some embodiments as illustrated in FIGS. 1 and 2A, the 5′ portion ofthe first primer can be a sequence not hybridizable to the targetsequence (under conditions in which the 3′ portion hybridizes to RNAtarget), e.g., a sequence forming a “tail” when the primer is hybridizedto a target. This “tail” sequence generally is incorporated into thefirst primer extension product (first strand cDNA), and the complementof this tail will be incorporated at the 3′ end of the second primerextension product (second strand EDNA). Accordingly, in someembodiments, the first primer is a mixture of first primers whichcomprise the same 5′ DNA portion and a multiplicity of 3′ DNA portionsselected to amplify a multiplicity (which can be small to very large) ofRNA sequences of interest. In other embodiments, the 5′ portion of thefirst primer can be hybridizable to the target RNA.

The second primer as illustrated in FIGS. 1 and 2A may comprise randomsequences, which are known in the art, and that are complementary tosequences of a plurality of the first cDNA strands produced. The 3′ endof the propromoter polynucleotide is preferably, but not necessarily,non-extendable. The 3′ portion of the PTO generally comprises a sequencethat is typically identical to the B sequence of the first primer 4.

Primer 2 as illustrated in FIGS. 1 and 2A can be, but is notnecessarily, composed of DNA and can comprise two sections(interchangeably called “portions” or “regions”). The 3′ portion ofprimer 2 can be selected for random priming of many, most and/or allpossible mRNA sequences in a biological sample. Random primers are knownin the art, for example, they have been used extensively in thepreparation of cDNA libraries using PCR-based procedures. In someembodiments, the hybridizable sequence of the second primer is designedbased on a known sequence of the desired binding site on a first strandcDNA. In other embodiments, the second primer comprises a strand switcholigonucleotide, described in U.S. Pat. Nos. 5,962,271 and 5,962,272,which is hybridizable to the Cap sequences present on mRNA and causesthe reverse transcriptase to switch from the mRNA template to the switcholigonucleotide, permitting generation of a second strand cDNA primed bythe “switch oligonucleotide”. Alternatively, a homopolymeric tail isadded to the 3′terminus of the first primer extension product, and thesecond primer comprises the complement of the homopolymeric tail.

In some embodiments, the second primer comprises DNA. In otherembodiments, the second primer consists of DNA. In other embodiments, asdescribed herein, the second primer is a fragment of the target RNA,with the fragment being generated by cleavage of the RNA target.

The 5′ portion of primer 2 can be a sequence which is not complementaryand not substantially hybridizable to a specific target sequence, i.e.,it would not hybridize (under conditions in which the 3′ portionhybridizes to RNA target) and would constitute a tail. The “tail”sequence would generally be incorporated into the second primerextension product.

A promoter template oligonucleotide, 3 (PTO), can be designed asfollows: the 3′ end of the propromoter polynucleotide is preferably, butnot necessarily, non-extendable. The 3′ portion of the PTO generallycomprises a sequence that is typically identical to the B sequence ofthe first primer 4. The 5′-most portion of the PTO is a promotersequence for a DNA-dependent RNA polymerase, which, as described above,is used in certain embodiments of the amplification methods of theinvention. Generally, the sequence between these two sections isdesigned for optimal transcription by the polymerase of choice. Criteriafor selection and design of this sequence are known in the art.

As illustrated in FIG. 1, one embodiment of the process of the linearamplification methods of the invention is as follows:

A) Formation of Double Stranded cDNA Substrate from Target RNA

-   -   1. Primer 4 binds to a target RNA by hybridization of its 3′ end        to the target to form complex I. The most commonly used        initiation site for binding of the first primer to generate        first strand cDNA is the 3′ end poly-A sequence of mRNA and the        immediate adjacent nucleotides. Binding of the primer to these        immediate adjacent nucleotides can be achieved by including        partially random sequences (other than sequence complementary to        poly-A sequence of a target mRNA) at the 3′ end of the primer.        Criteria for primer designs for this purpose are known in the        art, for example in the selection of primer sequences in the        generation of libraries (including cDNA libraries). In cases        where it is desired to produce libraries of sequences of a        pre-defined family of mRNA, such as for example preparation of        libraries from an immunoglobulin chain, the A sequence of the        first primer could comprise a sequence which is well preserved        in all members of the family of RNA from the immunoglobulin        chain.    -   2. Primer extension along the RNA strand of complex I is carried        out by an RNA-dependent DNA polymerase such as reverse        transcriptase (labeled “RT”), to form an RNA/DNA hybrid duplex.        RNase H degrades the RNA strand of the hybrid duplex to produce        a first cDNA strand (labeled “I”). The RNase H activity may be        supplied by the RNA-dependent DNA polymerase (such as reverse        transcriptase) or may be provided in a separate enzyme. Reverse        transcriptases useful for this method may or may not have RNase        H activity.    -   3. A second primer (labeled “5”) binds to II at a complementary        site to form complex III. In the case where a plurality of RNA        species are being amplified simultaneously, the second primers        could be random primers that bind to II at random complementary        sites of a plurality of cDNA species. When it is desired to        generate a library of a known mRNA family, such as for example a        library of a specific immunoglobulin chain, primer 5 may        comprise a sequence which is complementary to a well conserved        sequence in this family.    -   4. Primer extension along the first cDNA strand is carried out        by a DNA-dependent DNA polymerase to generate a double stranded        cDNA (labeled “IV”) comprising at one end a duplex of sequence B        (of the first primer) and its complement. The cDNAs are        substrates for amplification according to the methods of the        invention.    -   5. The double stranded products IV are denatured (for example,        by heat) to separate the two DNA strands. Various methods for        strand separation could be employed for carrying out the methods        of the invention. Strand V, which is of the same sense as the        input (target) RNA, is a substrate for amplification, and        comprises at its 5′ end the sequence of the second primer        (primer 5), and at its 3′ end the sequence complementary to        sequence B of the first primer (primer 4).        B) Linear Isothermal Amplification    -   1. A propromoter oligonucleotide (such as a PTO) binds to the 3′        end of strand V by hybridization of its 3′ end sequence B to its        complementary sequence at the 3′ end of V, to form complex VI.    -   2. A DNA-dependent DNA polymerase extends the 3′ end of the        sense cDNA strand (strand V) in complex VI to form the partial        duplex VII, comprising a double stranded RNA polymerase promoter        at one end.    -   3. An RNA polymerase binds to the double stranded promoter and        transcribes the cDNA strand of complex VII to produce multiple        copies of single stranded antisense RNA products, VIII. These        antisense RNA products can also serve as substrates for        exponential amplification.

One embodiment of the exponential amplification methods of the inventionis illustrated in FIGS. 2A-B. In this embodiment, subsequent to thegeneration of antisense RNA products in the linear amplification steps,the following steps are performed.

C) Exponential Isothermal Amplification

-   -   1. Primer 5 binds to the 3′ end of the RNA products VIII to form        complex IX.    -   2. Primer extension along the RNA strand is carried out by an        RNA-dependent DNA polymerase to form an RNA/DNA hybrid duplex,        X.    -   3. RNase H degrades the RNA strand of complex X to produce        single stranded DNA copies of the RNA products. A propromoter        polynucleotide such as a PTO hybridizes to the 3′ end of the        single stranded DNA to form complex XI.    -   4. The 3′ end of the DNA strand in complex XI is extended along        the propromoter polynucleotide (PTO) to form a partial duplex        XII, comprising a double stranded promoter sequence at one end.        This product is the same as VII, and is a substrate for RNA        polymerase for the generation of multiple copies of the RNA        products, as in step b(3) above, thus producing a cyclical        process for the exponential amplification of a target RNA.        Nucleic Acid Sequence Amplification Using a First Primer, a        Composite Primer, Denaturation and Strand Displacement

The invention provides methods of amplifying an RNA sequence of interestby using a first primer and a composite primer, denaturation, and stranddisplacement. Amplification can be achieved isothermally, though everystep is not necessarily conducted at the same temperature. Amplifiedproducts are single stranded DNA comprising sequences complementary tothe RNA sequence of interest in the target RNA.

A schematic description of one embodiment of the composite primer,second primer and strand displacement-based methods of the invention isgiven in FIGS. 3A and B. The methods involve the following steps: (a)formation of a second strand cDNA which is the same sense as the inputRNA (as described herein, and one embodiment of which is illustrated inFIG. 1); and (b) linear amplification of the complement of a secondstrand cDNA strand by primer extension from a composite primer along thesecond strand cDNA and strand displacement. See Kurn, U.S. Pat. No.6,251,639 B1.

The embodiment illustrated in FIGS. 3A and 3B employs threeoligonucleotides: a first primer which can be composed of two portions,a 3′ portion (labeled “A”); and a 5′ portion (labeled “B”), (labeled 1),used for formation of first strand cDNA; a second primer, used for theformation of the second strand cDNA (which may be an exogenously addedsecond primer or one or more fragments of target RNA that remainshybridized to the first strand cDNA following RNase H treatment); and acomposite primer used for linear isothermal amplification. The compositeprimer comprises a 5′ RNA portion and a DNA portion. The first andsecond primers used in this aspect of the invention may comprise anyfirst and second primer described herein (including a second primercomprising one or more fragments.

The composite primer illustrated in FIG. 3A and B comprises a sequencecapable of hybridizing to the second strand cDNA, and most oftencomprises a sequence hybridizable to the defined 3′-portion of thesecond strand cDNA (that is the complement of the first primersequence). The composite primer may additionally comprise sequences nothybridizable to the second strand cDNA under conditions which thecomposite primer hybridizes, such that a tail is formed.

As illustrated in FIGS. 3A and B, the composite primer comprises a DNAportion at its 3′ end, and an RNA portion at its 5′ end. As discussedherein, it is also possible to employ a composite primer in which the 3′DNA portion is followed, in the direction of its 5′, by an RNA portion,which is followed by a portion which is DNA. The length of each of thesesections is generally determined for maximum efficiency of theamplification. Only the two-portion (i.e., 3′-DNA-RNA-5′) compositeprimer is shown in FIGS. 3A and B.

As illustrated in FIGS. 3A and B, in one embodiment, the process of theamplification methods of the invention resulting in generation of DNAproducts comprising sequences complementary to an RNA sequence(s) ofinterest based on RNA is as follows:

A) Formation of a Single Stranded cDNA Substrate for Amplification Usinga Composite Primer

-   -   1. A primer binds to a target RNA by hybridization of its 3′ end        to the target.    -   2. Primer extension along the RNA strand is carried out by an        RNA-dependent DNA polymerase such as reverse transcriptase, to        form an RNA/DNA hybrid duplex. RNase H degrades the RNA strand        of the hybrid duplex to produce a first cDNA strand (labeled        “I”). The RNase H activity may be supplied by the RNA-dependent        DNA polymerase (such as reverse transcriptase) or may be        provided in a separate enzyme. Reverse transcriptases useful for        this method may or may not have RNase H activity.    -   3. Primer extension along the first cDNA strand is carried out        by a DNA-dependent DNA polymerase (not illustrated in FIG. 3A)        to generate a double stranded cDNA, forming a complex of first        and second strand cDNAs (wherein the second strand cDNA        comprises a 3′ end portion that is the complement of the first        primer sequence), as shown in FIG. 3A.    -   4. The complex of first and second strand cDNAs is then        denatured to form single stranded first strand cDNA and second        strand cDNA, as shown in FIG. 3B. The single stranded second        strand cDNA is the substrate for isothermal amplification using        a composite primer and strand displacement as follows.        B) Generation of Bouble Stranded cDNA Comprising an RNA/DNA        Heteroduplex    -   1. A composite primer comprising a 5′-RNA portion and a DNA        portion hybridizes to the 3′-portion of the second strand cDNA,        generally to the 3′-portion of the second strand cDNA, and is        extended along the second strand cDNA by a DNA polymerase to        form a double stranded complex comprising an RNA/DNA hybrid        portion at one end of the complex.        C) Isothermal Linear Amplification    -   1. An agent, such as an enzyme, which cleaves RNA from an        RNA/DNA hybrid (such as RNase H) cleaves RNA sequence from the        hybrid, leaving a sequence on the second strand cDNA available        for binding by another composite primer.    -   2. Composite primer binds by hybridization of the RNA portion to        the single stranded DNA end on the second strand cDNA, which is        complementary to it. The 3′ DNA sequence of primer is not        hybridized.    -   3. The 3′ end of bound composite primer, and the 5′ end of the        DNA strand immediately upstream are the same, and would compete        for hybridization to the opposite strand. Without wishing to be        bound by theory, the high affinity of the DNA polymerase to        hybridized 3′ end of a primer would be expected to push the        equilibrium of the two competing structures towards        hybridization of the 3′ end of the new primer and displacement        of the 5′ end of the previous primer extension product. Primer        extension along the second strand (sense) cDNA strand results in        displacement of the previous second strand cDNA, and formation        of a double stranded cDNA having, at one end, an RNA/DNA hybrid        composed of sequence B and its complement    -   4. The RNA segment of the hybrid is degraded by agent, such as        RNase H, which results in the formation of a single stranded 3′        end to which a new composite primer can be bound by its 5′        portion.    -   5. The process of hybridization of the 3′ end sequence of the        bound composite primer, by displacement of the 5′-most end of        the previous primer extension product in the duplex, primer        extension and displacement of the previous product continues,        and results in the accumulation of multiple copies of anti-sense        single stranded DNA products.

In some embodiments, subsequent to the generation of antisense RNAproducts in the linear amplification steps, the following steps areperformed:

D) Exponential Isothermal Amplification

-   -   1. A propromoter oligonucleotide (such as a PTO) binds to the 3′        end of anti-sense single stranded DNA products.    -   2. Primer extension along the RNA strand is carried out by an        RNA-dependent DNA polymerase to form an RNA/DNA hybrid duplex.        This product is the same is a substrate for RNA polymerase for        the generation of multiple copies of sense RNA products. As        described herein, extension of the propromoter polynucleotide        may or may not be required to effect creation of a propromoter        for transcription.        Components and Reaction Conditions Used in the Methods of the        Invention        Template Nucleic Acid

The RNA target to be amplified includes RNAs from any source in purifiedor unpurified form, which can be RNA such as total RNA, tRNA, mRNA,rRNA, mitochondrial RNA, chloroplast RNA, DNA-RNA hybrids, or mixturesthereof, from any source and/or species, including human, animals,plants, and microorganisms such as bacteria, yeasts, viruses, viroids,molds, fungi, plants, and fragments thereof. RNAs can be obtained andpurified using standard techniques in the art. Amplification of a DNAtarget would require initial transcription of the DNA target into RNAform, which can be achieved by techniques (such as expression systems)known in the art. Amplification of a DNA-RNA hybrid would requiredenaturation of the hybrid to obtain a ssRNA, or denaturation followedby transcription of the DNA strand to obtain an RNA. The target RNA canbe only a minor fraction of a complex mixture such as a biologicalsample and can be obtained from various biological material byprocedures well known in the art.

The target RNA can be known or unknown and may contain more than onedesired specific nucleic acid sequence of interest, each of which may bethe same or different from each other. Therefore, the amplificationprocess is useful not only for producing large amounts of one specificnucleic acid sequence, but also for amplifying simultaneously more thanone different specific nucleic acid sequence located on the same ordifferent nucleic acid molecules.

The initial step of the amplification of a target RNA sequence isrendering the target single stranded. Denaturation may be carried out toremove secondary structure present in a RNA target molecule. Thedenaturation step may be thermal denaturation or any other method knownin the art.

First Primer

The first primer is a primer that comprises a sequence (which may or maynot be the whole of the primer) that is hybridizable (under a given setof conditions) to the target RNA. This sequence can be based on aspecific sequence of the target, or a random sequence (in someembodiments, the first primer is a random primer). It can also be basedon a general, more universal sequence known to be present in an RNAspecies of interest, such as the poly-A sequence found in mRNA. In someembodiments, the primer may comprise a sequence complementary to apoly-A sequence, and may further comprise a random sequence 3′ to saidsequence complementary to a poly-A sequence. In some embodiments, theprimer comprises a sequence, preferably at the 5′ end, that is nothybridizable (under a given set of conditions) to a target RNA. Inaddition, the sequence that is capable of hybridizing to the target RNAmay comprise a sequence complementary to the poly-A tail of mRNA, andmay further comprise additional random sequences (generally notcomplementary to a poly-A sequence) at the 3′ end of the 3′ portion.

Random primers are well known in the art, and include at least thefollowing: primers hybridizable to two or more sequences in a sample;and primers comprising poly-T sequences that are hybridizable to amultiplicity of RNAs in a sample (such as all mRNA). For convenience, asingle random composite primer is discussed above. However, it isunderstood that the term “random primer” can refer to a primer that is amember of a population of primers which are collectively designed to adesired and/or significant population of target sequences.

It is also understood that the amplification of a plurality of mRNAspecies in a single reaction mixture may, but not necessarily, employ amultiplicity, or a large multiplicity of primers. Thus, the inventioncontemplates the use of a multiplicity of different composite primers(random or non-random) when amplifying a plurality of mRNA species in asingle reaction mixture.

To achieve hybridization to a target nucleic acid (which, as is wellknown and understood in the art, depends on other factors such as, forexample, ionic strength and temperature), the sequence of the primerthat is hybridizable to the target RNA is preferably of at least about60%, more preferably at least about 75%, even more preferably at leastabout 90%, and most preferably at least about 95% complementarily to thetarget nucleic acid.

In some embodiments, the first primer comprises a 5′ sequence (whichgenerally includes the 5′ most nucleotide) the complement of which ishybridizable by a propromoter polynucleotide. This sequence enables thecreation of a defined end sequence for the 5′ end of the first primerextension product (and thus, subsequently the 3′ end of the second/thirdprimer extension product). Having a defined end sequence is particularlyadvantageous with respect to hybridization of a propromoterpolynucleotide to the 3′ end of the second and third primer extensionproducts in subsequent steps. Thus, in these embodiments, the firstprimer comprises a sequence that when introduced into the amplificationsteps of the methods of the invention results in generation of anintermediate polynucleotide to which a propromoter polynucleotide canhybridize. In some of these embodiments, the 5′ sequence the complementof which is hybridizable by a propromoter polynucleotide is hybridizable(under a given set of conditions) to a target RNA when the primer ishybridized to the target RNA. In other embodiments, the 5′ sequence thecomplement of which is hybridizable by a propomoter polynucleotide isnot hybridizable (under a given set of conditions) to a target RNA whenthe primer is hybridized to the target RNA (thus constituting a tailwhen the 3′ sequence of the primer is hybridized to the target).

In one embodiment, the first primer comprises DNA. In anotherembodiment, the first primer comprises RNA. In yet another embodiment,the first primer comprises DNA and RNA.

Second Primer

The second primer in the methods of the invention comprises a sequence(which may or may not be the whole of the primer) that is hybridizable(under a given set of conditions) to a first primer extension product ata site on the first primer extension product such that the second primerextension product would include the RNA sequence of interest. In someembodiments, the hybridizable sequence of the second primer is designedbased on a known sequence of the desired binding site on a first primerextension product. In other embodiments, the hybridizable sequence isbased on random sequences known in the art to be suitable for randompriming of a plurality of RNA species. In some embodiments, a secondprimer comprises a sequence, preferably at the 5′ end, that is nothybridizable (under a given set of conditions) to a first primerextension product. In other embodiments, the second primer comprises astrand switch oligonucleotide, described in U.S. Pat. Nos. 5,962,271 and5,962,272, which is hybridizable to the Cap sequences present on mRNAand causes the reverse transcriptase to switch from the mRNA template tothe switch oligonucleotide, permitting generation of a second strandcDNA primed by the “switch oligonucleotide”. Alternatively, ahomopolymeric tail is added to the 3′ terminus of the first primerextension product, and the second primer comprises the complement of thehomopolymeric tail.

In one embodiment, the second primer comprises DNA. In anotherembodiment, the second primer consists of DNA. In another embodiment,the second primer comprises RNA. In yet another embodiment, the secondprimer comprises DNA and RNA.

In some embodiments, the second primer is provided by self priming (forexample, by a hairpin loop) at the 3′ end of the first primer extensionproduct. In these embodiments, a sequence at the 3′ end of the firstprimer extension product hybridizes to another sequence in the firstprimer extension product, for example as described in U.S. Pat. No.6,132,997. In these embodiments, said sequence at the 3′ of the firstprimer extension product is generally cleaved (for example, with an S1nuclease) following its hybridization to the first primer extensionproduct and/or its extension along the first primer extension product.See U.S. Pat. No. 6,132,997.

In some embodiments, the second primer is provided by a target RNAfragment. Such a target RNA fragment can be generated as a result ofincomplete degradation of a target RNA in a complex of target RNA andfirst primer extension product by an enzyme that cleaves RNA in anRNA/DNA hybrid, such that one or more RNA fragments remain bound to thefirst primer extension product.

To achieve hybridization to a first primer extension product (which, asis well known and understood in the art, depends on other factors suchas, for example, ionic strength and temperature), the sequence of thesecond primer that is hybridizable to the first primer extension productis preferably of at least about 60%, more preferably at least about 75%,even more preferably at least about 90%, and most preferably at leastabout 95% complementarity to the first primer extension product.

Third Primer

The third primer in the methods of the invention comprises a sequence(which may or may not be the whole of the primer) that is hybridizable(under a given set of conditions) to the RNA transcript generated fromthe second primer extension product (as the template) at a site on theRNA transcript such that the third primer extension product wouldinclude the RNA sequence of interest, if present. In some embodiments,the hybridizable sequence of the third primer is designed based on aknown sequence of the desired binding site on an RNA transcript. Inother embodiments, the hybridizable sequence is based on randomsequences known in the art to be suitable for random priming of aplurality of RNA species. In some embodiments, the third primercomprises a sequence, preferably at the 5′ end, that is not hybridizable(under a given set of conditions) to an RNA transcript.

To achieve hybridization to an RNA transcript (which, as is well knownand understood in the art, depends on other factors such as, forexample, ionic strength and temperature), the sequence of the thirdprimer that is hybridizable to the RNA transcript is preferably of atleast about 60%, more preferably at least about 75%, even morepreferably at least about 90%, and most preferably at least about 95%complementarity to the RNA transcript.

In one embodiment, the third primer comprises DNA. In anotherembodiment, the third primer comprises RNA. In yet another embodiment,the third primer comprises DNA and RNA.

In some embodiments, the third primer is provided by self priming (forexample, by a hairpin loop) at the 3′ end of an RNA transcript. In theseembodiments, a sequence at the 3′ end of the RNA transcript hybridizesto another sequence in the RNA transcript itself. In these embodiments,said sequence at the 3′ of the RNA transcript is generally cleavedfollowing its hybridization to the RNA transcript and/or its extensionalong the RNA transcript.

Composite Primer

In some embodiments, the methods of the invention employ a compositeprimer that is composed of RNA and DNA portions. The composite primer isdesigned such that subsequent displacement of the primer extensionproduct by binding of a new (additional) composite primer and theextension of the new primer by the polymerase can be achieved. Inaddition, cleavage of the RNA portion of the primer extension productleads to generation of amplification product which is not a substratefor amplification by the composite primer.

The composite primer illustrated in FIG. 3 comprises sequences capableof hybridizing to the second strand cDNA, and most often comprisessequences hybridizable to the defined 3′-portion of the second strandcDNA (that is the complement of the first primer sequence). Thecomposite primer may comprise all or a portion of the sequence of thefirst primer. The composite primer may additionally comprise sequencesnot hybridizable to the second strand cDNA such that a tail is formed.

It is also understood that the amplification of a plurality of mRNAspecies in a single reaction mixture may, but not necessarily, employ amultiplicity, or a large multiplicity of primers. Thus, the inventioncontemplates the use of a multiplicity of different composite primers(random or non-random) when amplifying a plurality of mRNA species in asingle reaction mixture.

A composite primer comprises at least one RNA portion that is capable of(a) binding (hybridizing) to a sequence on the second strand cDNAproduct independent of hybridization of the DNA portion(s) to a sequenceon the same extension product; and (b) being cleaved with a ribonucleasewhen hybridized to the second strand cDNA. The composite primers bind tothe second strand cDNA to form a partial heteroduplex in which only theRNA portion of the primer is cleaved upon contact with a ribonucleasesuch as RNase H, while the second strand cDNA remains intact, thusenabling annealing of another composite primer.

The composite primers also comprise a 3′ DNA portion that is capable ofhybridization to a sequence on the second strand cDNA such that itshybridization to the cDNA is favored over that of the nucleic acidstrand that is displaced from the second strand cDNA by the DNApolymerase. Such primers can be rationally designed based on well knownfactors that influence nucleic acid binding affinity, such as sequencelength and/or identity, as well as hybridization conditions. In apreferred embodiment, hybridization of the 3′ DNA portion of thecomposite primer to its complementary sequence in the second strand cDNAis favored over the hybridization of the homologous sequence in the 5′end of the displaced strand to the second strand cDNA.

Generation of primers suitable for extension by polymerization is wellknown in the art, such as described in PCT Pub. No. WO 99/42618 (andreferences cited therein). The composite primer comprises a combinationof RNA and DNA (see definition above), with the 3′-end nucleotide beinga nucleotide suitable for nucleic acid extension. The 3′-end nucleotidecan be any nucleotide or analog that when present in a primer, isextendable by a DNA polymerase. Generally, the 3′-end nucleotide has a3′-OH. Suitable primers include those that comprise at least one portionof RNA and at least one portion of DNA. For example, composite primerscan comprise a 5′-RNA portion and a 3′-DNA portion (in which the RNAportion is adjacent to the 3′-DNA portion); or 5′- and 3′-DNA portionswith an intervening RNA portion. Accordingly, in one embodiment, thecomposite primer comprises a 5′ RNA portion and a 3′-DNA portion,preferably wherein the RNA portion is adjacent to the 3′-DNA portion. Inanother embodiment, the composite primer comprises 5′ and 3′-DNAportions with at least one intervening RNA portion (i.e., an RNA portionbetween the two DNA portions). In yet another embodiment, the compositeprimer of the invention comprises a 3′-DNA portion and at least oneintervening RNA portion (i.e., an RNA portion between DNA portions).

The length of an RNA portion in a composite primer comprising a 3′-DNAportion and an RNA portion can be preferably from about 1 to about 50,more preferably from about 3 to about 20, even more preferably fromabout 4 to about 15, and most preferably from about 5 to about 10nucleotides. In some embodiments of a composite primer comprising a3′-DNA portion and an RNA portion, an RNA portion can be at least aboutany of 1, 3, 4, 5 nucleotides, with an upper limit of about any of 10,15, 20, 25, 3, 50 nucleotides.

The length of the 5′-RNA portion in a composite primer comprising a5′-RNA portion and a 3′-DNA portion can be preferably from about 3 toabout 50 nucleotides, more preferably from about 5 to about 20nucleotides, even more preferably from about 7 to about 18 nucleotides,preferably from about 8 to about 17 nucleotides, and most preferablyfrom about 10 to about 15 nucleotides. In other embodiments of acomposite primer comprising a 5′-RNA portion and a 3′-DNA portion, the5′-RNA portion can be at least about any of 3, 5, 7, 8, 10 nucleotides,with an upper limit of about any of 15, 17, 18, 20, 50 nucleotides.

In embodiments of a composite primer comprising a 5′-RNA portion and a3′-DNA portion further comprising non-5′-RNA portion(s), a non-5′-RNAportion can be preferably from about 1 to about 7 nucleotides, morepreferably from about 2 to about 6 nucleotides, and most preferably fromabout 3 to about 5 nucleotides. In certain embodiments of a compositeprimer comprising a 5′-RNA portion and a 3′-DNA portion furthercomprising non-5′-RNA portion(s), a non-5′-RNA portion can be at leastabout any of 1, 2, 3, 5, with an upper limit of about any of 5, 6, 7, 10nucleotides.

In embodiments of a composite primer comprising a 5′-RNA portion and a3′-DNA portion, in which the 5′-RNA portion is adjacent to the 3′-DNAportion, the length of the 5′-RNA portion can be preferably from about 3to about 50 nucleotides, more preferably from about 5 to about 20nucleotides, even more preferably from about 7 to about 18 nucleotides,preferably from about 8 to about 17 nucleotides, and most preferablyfrom about 10 to about 15 nucleotides. In certain embodiments of acomposite primer comprising a 5′-RNA portion and a 3′-DNA portion, inwhich the 5′-RNA portion is adjacent to the 3′-DNA portion, the 5′-RNAportion can be at least about any of 3, 5, 7, 8, 10 nucleotides, with anupper limit of about any of 15, 17, 18, 20, 50 nucleotides.

The length of an intervening RNA portion in a composite primercomprising 5′- and 3′-DNA portions with at least one intervening RNAportion can be preferably from about 1 to about 7 nucleotides, morepreferably from about 2 to about 6 nucleotides, and most preferably fromabout 3 to about 5 nucleotides. In some embodiments of a compositeprimer comprising 5′- and 3′-DNA portions with at least one interveningRNA portion, an intervening RNA portion can be at least about any of 1,2, 3, 5 nucleotides, with an upper limit of about any of 5, 6, 7, 10nucleotides. The length of an intervening RNA portion in a compositeprimer comprising a 3′-DNA portion and at least one intervening RNAportion can be preferably from about 1 to about 7 nucleotides, morepreferably from about 2 to about 6 nucleotides, and most preferably fromabout 3 to about 5 nucleotides. In some embodiments of a compositeprimer comprising a 3′-DNA portion and at least one intervening RNAportion, an intervening RNA portion can be at least about any of 1, 2,3, 5 nucleotides, with an upper limit of about any of 5, 6, 7, 10nucleotides. In a composite primer comprising a 3′-DNA portion and atleast one intervening RNA portion, further comprising a 5′-RNA portion,the 5′-RNA portion can be preferably from about 3 to about 25nucleotides, more preferably from about 5 to about 20 nucleotides, evenmore preferably from about 7 to about 18 nucleotides, preferably fromabout 8 to about 17 nucleotides, and most preferably from about 10 toabout 15 nucleotides. In some embodiments of a composite primercomprising a 3′-DNA portion and at least one intervening RNA portion,further comprising a 5′-RNA portion, the 5′-RNA portion can be at leastabout any of 3, 5, 7, 8, 10 nucleotides, with an upper limit of aboutany of 15, 17, 18, 20 nucleotides.

The length of the 3′-DNA portion in a composite primer comprising a3′-DNA portion and an RNA portion can be preferably from about 1 toabout 20, more preferably from about 3 to about 18, even more preferablyfrom about 5 to about 15, and most preferably from about 7 to about 12nucleotides. In some embodiments of a composite primer comprising a3′-DNA portion and an RNA portion, the 3′-DNA portion can be at leastabout any of 1, 3, 5, 7, 10 nucleotides, with an upper limit of aboutany of 10, 12, 15, 18, 20, 22 nucleotides.

The length of the 3′-DNA portion in a composite primer comprising a5′-RNA portion and a 3′-DNA portion can be preferably from about 1 toabout 20 nucleotides, more preferably from about 3 to about 18nucleotides, even more preferably from about 5 to about 15 nucleotides,and most preferably from about 7 to about 12 nucleotides. In someembodiments of a composite primer comprising a 5′-RNA portion and a3′-DNA portion, the 3′ DNA portion can be at least about any of 1, 3, 5,7, 10 nucleotides, with an upper limit of about any of 10, 12, 15, 18,20, 22 nucleotides.

In embodiments of a composite primer comprising a 5′-RNA portion and a3′-DNA portion, further comprising non-3′-DNA portion(s), a non-3′-DNAportion can be preferably from about 1 to about 10 nucleotides, morepreferably from about 2 to about 8 nucleotides, and most preferably fromabout 3 to about 6 nucleotides. In some embodiments of a compositeprimer comprising a 5′-RNA portion and a 3′-DNA portion, furthercomprising non-3′-DNA portion(s), a non-3′-DNA portion can be at leastabout any of 1, 2, 3, 5 nucleotides, with an upper limit of about any of6, 8, 10, 12 nucleotides.

In embodiments of a composite primer comprising a 5′-RNA portion and a3′-DNA portion in which the 5′-RNA portion is adjacent to the 3′-DNAportion, the length of the 3′-DNA portion can be preferably from about 1to about 20 nucleotides, more preferably from about 3 to about 18nucleotides, even more preferably from about 5 to about 15 nucleotides,and most preferably from about 7 to about 12 nucleotides. In certainembodiments of the primer comprising a 5′-RNA portion and a 3′-DNAportion in which the 5′-RNA portion is adjacent to the 3′-DNA portion,the 3′-DNA portion can be at least about any of 1, 3, 5, 7, 10nucleotides, with an upper limit of about any of 10, 12, 15, 18, 20, 22nucleotides.

The length of a non-3′-DNA portion in a composite primer comprising 5′-and 3′-DNA portions with at least one intervening RNA portion can bepreferably from about 1 to about 10 nucleotides, more preferably fromabout 2 to about 8 nucleotides, and most preferably from about 3 toabout 6 nucleotides. In some embodiments of a primer comprising 5′- and3′-DNA portions with at least one intervening RNA portion, a non-3′-DNAportion can be at least about any of 1, 2, 3, 5 nucleotides, with anupper limit of about any of 6, 8, 10, 12 nucleotides.

The length of the 3′-DNA portion in a composite primer comprising 5′-and 3′-DNA portions with at least one intervening RNA portion can bepreferably from about 1 to about 20 nucleotides, more preferably fromabout 3 to about 18 nucleotides, even more preferably from about 5 toabout 15 nucleotides, and most preferably from about 7 to about 12nucleotides. In some embodiments of a composite primer comprising 5′-and 3′-DNA portions with at least one intervening RNA portion, the3′-DNA portion can be at least about any of 1, 3, 5, 7, 10 nucleotides,with an upper limit of about any of 10, 12, 15, 18, 20, 22 nucleotides.

The length of a non-3′-DNA portion (i.e., any DNA portion other than the3′-DNA portion) in a composite primer comprising a 3′-DNA portion and atleast one intervening RNA portion can be preferably from about 1 toabout 10 nucleotides, more preferably from about 2 to about 8nucleotides, and most preferably from about 3 to about 6 nucleotides. Insome embodiments of a composite primer comprising a 3′-DNA portion andat least one intervening RNA portion, a non-3′-DNA portion can be atleast about any of 1, 3, 5, 7, 10 nucleotides, with an upper limit ofabout any of 6, 8, 10, 12 nucleotides. The length of the 3′-DNA portionin a composite primer comprising a 3′-DNA portion and at least oneintervening RNA portion can be preferably from about 1 to about 20nucleotides, more preferably from about 3 to about 18 nucleotides, evenmore preferably from about 5 to about 15 nucleotides, and mostpreferably from about 7 to about 12 nucleotides. In some embodiments ofa composite primer comprising a 3′-DNA portion and at least oneintervening RNA portion, the 3′-DNA portion can be at least about any of1, 3, 5, 7, 10 nucleotides, with an upper limit of about any of 10, 12,15, 18, 20, 22 nucleotides. It is understood that the lengths for thevarious portions can be greater or less, as appropriate under thereaction conditions of the methods of this invention.

In some embodiments, the 5′-DNA portion of a composite primer includesthe 5′-most nucleotide of the primer. In some embodiments, the 5′-RNAportion of a composite primer includes the 5′ most nucleotide of theprimer. In other embodiments, the 3′-DNA portion of a composite primerincludes the 3′ most nucleotide of the primer. In other embodiments, the3′-DNA portion is adjacent to the 5′-RNA portion and includes the 3′most nucleotide of the primer (and the 5′-RNA portion includes the 5′most nucleotide of the primer).

The total length of the composite primer can be preferably from about 10to about 50 nucleotides, more preferably from about 15 to about 30nucleotides, and most preferably from about 20 to about 25 nucleotides.In some embodiments, the length can be at least about any of 10, 15, 20,25 nucleotides, with an upper limit of about any of 25, 30, 50, 60nucleotides. It is understood that the length can be greater or less, asappropriate under the reaction conditions of the methods of thisinvention.

To achieve hybridization to a target nucleic acid (which, as is wellknown and understood in the art, depends on other factors such as, forexample, ionic strength and temperature), the portion of the primer thatis hybridizable to the target RNA is preferably of at least about 60%,more preferably at least about 75%, even more preferably at least about90%, and most preferably at least about 95% complementarity to thetarget nucleic acid.

Polynucleotide Comprising a Propromoter and a Region Which Hybridizes toa Primer Extension Product

The methods of the invention employ a propromoter polynucleotidecomprising a propromoter and a region which hybridizes to a primerextension product. In some embodiments, the propromoter polynucleotideis provided as a PTO, as described in greater detail below.

Propromoter Template Oligonucleotide

In some embodiments, the methods employ a promoter sequence fortranscription which is provided by a propromoter templateoligonucleotide (PTO).

A PTO for use in the methods and compositions of the invention is asingle-stranded polynucleotide, generally DNA, comprising a propromotersequence that is designed for formation of a double stranded promoter ofan RNA polymerase, and a portion capable of hybridizing to the 3′ end ofa primer extension product. In a preferred embodiment, the propromotersequence is located in the 5′ portion of the oligonucleotide and thehybridizing sequence is located in the 3′ portion of theoligonucleotide. In one embodiment, and most typically, the promoter andhybridizing sequences are different sequences. In another embodiment,the promoter and hybridizing sequences overlap in sequence identity. Inyet another embodiment, the promoter and hybridizing sequences are thesame sequence, and thus are in the same location on the PTO. In theembodiments wherein hybridization of the PTO to the primer extensionproduct results in a duplex comprising an overhang (the 5′ end of thePTO that does not hybridize to the primer extension product, typicallycomprising all or part of the propromoter sequence), DNA polymerasefills in the overhang to create a double stranded promoter capable ofeffecting transcription by a suitable RNA polymerase.

Promoter sequences that allow transcription of a template DNA are knownin the art and have been discussed above. Preferably, the promotersequence is selected to provide optimal transcriptional activity of theparticular RNA polymerase used. Criteria for such selection, i.e., aparticular promoter sequence particularly favored by a particular RNApolymerase, is also known in the art. For example, the sequences of thepromoters for transcription by T7 DNA dependent RNA polymerase and SP6are known in the art. The promoter sequence can be from a prokaryotic oreukaryotic source.

In some embodiments, the PTO comprises an intervening sequence between apropromoter sequence and a portion capable of hybridizing to the 3′ endof the primer extension product. Suitable length of the interveningsequence can be empirically determined, and can be at least about 1, 2,4, 6, 8, 10, 12, 15 nucleotides. Suitable sequence identity of theintervening sequence can also be empirically determined, and thesequence is designed to preferably, but not necessarily, enhance degreeof amplification as compared to omission of the sequence. In oneembodiment, the intervening sequence is a sequence that is designed toprovide for enhanced, or more optimal, transcription by the RNApolymerase used. Generally, the sequence is not related (i.e., it doesnot substantially hybridize) to a primer extension product. More optimaltranscription occurs when transcriptional activity of the polymerasefrom a promoter that is operatively linked to said sequence is greaterthan from a promoter that is not so linked. The sequence requirementsfor optimal transcription are generally known in the art as previouslydescribed for various DNA dependent RNA polymerases, such as in U.S.Pat. Nos. 5,766,849 and 5,654,142, and can also be empiricallydetermined.

In another embodiment, the PTO comprises a sequence that is 5′ to thepropromoter sequence, i.e., the PTO comprises additional nucleotides(which may or may not be transcriptional regulatory sequences) located5′ to the propromoter sequence. Generally, but not necessarily, thesequence is not hybridizable (under a given set of conditions) to theprimer extension product.

In one embodiment, the PTO cannot function efficiently as a primer fornucleic acid extension. Techniques for blocking the primer function ofthe PTO include any that prevent addition of nucleotides to the 3′ endof the PTO by a DNA polymerase. Such techniques are known in the art,including, for example, substitution or modification of the 3′ hydroxylgroup, or incorporation of a modified nucleotide, such as adideoxynucleotide, in the 3′-most position of the PTO that is notcapable of anchoring addition of nucleotides by a DNA polymerase. It ispossible to block the 3′ end using a label, or a small molecule which isa member of a specific binding pair, such as biotin. It is also possibleto render the 3′ end non-extendable by addition of nucleotides whichcannot hybridize to a primer extension product, either due tonon-complementarity or due to structural modifications which do notsupport hydrogen bonding. In other embodiments, the PTO is not blocked.

The length of the portion of the PTO that hybridizes to a primerextension product of interest is preferably from about 5 to about 50nucleotides, more preferably from about 10 to about 40 nucleotides, evenmore preferably from about 15 to about 35 nucleotides, and mostpreferably from about 20 to 30 nucleotides. In some embodiments, thehybridizing portion is at least about any of the following: 3, 5, 10,15, 20; and less than about any of the following: 30, 40, 50, 60. Thecomplementarity of the hybridizing portion is preferably at least about25%, more preferably at least about 50%, even more preferably at leastabout 75%, and most preferably at least about 90%, to its intendedbinding sequence on the primer extension product of interest.

DNA Polymerase, Ribonuclease and RNA Polymerase

The amplification methods of the invention employ the following enzymes:an RNA-dependent DNA polymerase, a DNA-dependent DNA polymerase, aribonuclease such as RNase H, and a DNA-dependent RNA polymerase. One ormore of these activities may be found and used in a single enzyme. Forexample, RNase H activity may be supplied by an RNA-dependent DNApolymerase (such as reverse transcriptase) or may be provided in aseparate enzyme. Reverse transcriptases useful for this method may ormay not have RNase H activity.

One aspect of the invention is the formation of double stranded cDNAfrom a primer-RNA complex. This process generally utilizes the enzymaticactivities of an RNA-dependent DNA polymerase, a DNA-dependent DNApolymerase and a ribonuclease activity.

RNA-dependent DNA polymerases for use in the methods and compositions ofthe invention are capable of effecting extension of a primer accordingto the methods of the invention. Accordingly, a preferred RNA-dependentRNA polymerase is one that is capable of extending a nucleic acid primeralong a nucleic acid template that is comprised at least predominantlyof ribonucleotides. Suitable RNA-dependent DNA polymerases for use inthe methods and compositions of the invention include reversetranscriptase. Many reverse transcriptases, such as those from avianmyeoloblastosis virus (AMV-RT), and Moloney murine leukemia virus(MMLV-RT) comprise more than one activity (for example, polymeraseactivity and ribonuclease activity) and can function in the formation ofthe double stranded cDNA molecules. However, in some instances, it ispreferable to employ a reverse transcriptase which lacks the RNase Hactivity. Reverse transcriptase devoid of RNase H activity are known inthe art, including those comprising a mutation of the wild type reversetranscriptase where the mutation eliminates the RNase H activity. Inthese cases, the addition of an RNase H from other sources, such as thatisolated from E. coli, can be employed for the formation of the doublestranded cDNA.

DNA-dependent DNA polymerases for use in the methods and compositions ofthe invention are capable of effecting extension of a primer accordingto the methods of the invention. Accordingly, a preferred polymerase isone that is capable of extending a nucleic acid primer along a nucleicacid template that is comprised at least predominantly ofdeoxynucleotides. The formation of the double stranded cDNA can becarried out by reverse transcriptase which comprises both RNA-dependentDNA polymerase and DNA-dependent DNA polymerase activities. Preferably,the DNA polymerase has high affinity for binding at the 3′-end of anoligonucleotide hybridized to a nucleic acid strand. Preferably, the DNApolymerase does not possess substantial nicking activity. Preferably,the polymerase has little or no 5′->3′ exonuclease activity so as tominimize degradation of primer, or primer extension polynucleotides.Generally, this exonuclease activity is dependent on factors such as pH,salt concentration, whether the template is double stranded or singlestranded, and so forth, all of which are familiar to one skilled in theart. Mutant DNA polymerases in which the 5′->3′ exonuclease activity hasbeen deleted, are known in the art and are suitable for theamplification methods described herein. Preferably, the DNA polymerasehas little to no proofreading activity.

Suitable DNA polymerases for use in the methods and compositions of theinvention include those disclosed in U.S. Pat. Nos. 5,648,211 and5,744,312, which include exo⁻ Vent (New England Biolabs), exo⁻ Deep Vent(New England Biolabs), Bst (BioRad), exo⁻ Pfu (Stratagene), Bca(Panvera), sequencing grade Taq (Promega), and thermostable DNApolymerases from thermoanaerobacter thermohydrosulfuiricus.

An agent that cleaves RNA in an RNA/DNA hybrid (e.g. ribonuclease) isused in the methods and compositions of the invention. Preferably, theagent, which can be ribonuclease, cleaves ribonucleotides regardless ofthe identity and type of nucleotides adjacent to the ribonucleotide tobe cleaved. It is preferred that the agent (e.g. ribonuclease) cleavesindependent of sequence identity. Examples of suitable ribonucleases forthe methods and compositions of the invention are well known in the art,including ribonuclease H (RNase H), including Hybridase.

The DNA-dependent RNA polymerase for use in the methods and compositionsof the invention are known in the art. Either eukaryotic or prokaryoticpolymerases may be used. Examples include T7, T3 and SP6 RNApolymerases. Generally, the RNA polymerase selected is capable oftranscribing from the promoter sequence provided by the propromoterpolynucleotides as described herein. Generally, the RNA polymerase is aDNA-dependent polymerase, which is preferably capable of transcribingfrom a single stranded DNA template so long as the promoter region isdouble stranded.

In general, the enzymes used included in the methods and compositions ofthe invention should not produce substantial degradation of the nucleicacid components of said methods and compositions.

Reaction Conditions and Detection

Appropriate reaction media and conditions for carrying out the methodsof the invention are those that permit nucleic acid amplificationaccording to the methods of the invention. Such media and conditions areknown to persons of skill in the art, and are described in variouspublications, such as U.S. Pat. Nos. 5,554,516; 5,716,785; 5,130,238;5,194,370; 6,090,591; 5,409,818; 5,554,517; 5,169,766; 5,480,784;5,399,491; 5,679,512; and PCT Pub. No. WO99/42618. For example, a buffermay be Tris buffer, although other buffers can also be used as long asthe buffer components are non-inhibitory to enzyme components of themethods of the invention. The pH is preferably from about 5 to about 11,more preferably from about 6 to about 10, even more preferably fromabout 7 to about 9, and most preferably from about 7.5 to about 8.5. Thereaction medium can also include bivalent metal ions such as Mg²⁺ orMn²⁺, at a final concentration of free ions that is within the range offrom about 0.01 to about 15 mM, and most preferably from about 1 to 10mM. The reaction medium can also include other salts, such as KCl orNaCl, that contribute to the total ionic strength of the medium. Forexample, the range of a salt such as KCl is preferably from about 0 toabout 125 mM, more preferably from about 0 to about 100 mM, and mostpreferably from about 0 to about 75 mM. The reaction medium can furtherinclude additives that could affect performance of the amplificationreactions, but that are not integral to the activity of the enzymecomponents of the methods. Such additives include proteins such as BSA,single stranded binding protein (for example, T4 gene 32 protein), andnon-ionic detergents such as NP40 or Triton. Reagents, such as DTT, thatare capable of maintaining enzyme activities can also be included. Suchreagents are known in the art. Where appropriate, an RNase inhibitor(such as Rnasin) that does not inhibit the activity of the RNaseemployed in the method can also be included. Any aspect of the methodsof the invention can occur at the same or varying temperatures.Preferably, the amplification reactions (particularly, primer extensionand transcription; and generally not the step of denaturing the complexof first and second primer extension products) are performedisothermally, which substantially avoids the cumbersome thermocyclingprocess. The amplification reaction is carried out at a temperature thatpermits hybridization of the oligonucleotides (primer, and/or PTO) ofthe invention to the template polynucleotide and that does notsubstantially inhibit the activity of the enzymes employed. Thetemperature can be in the range of preferably about 25° C. to about 85°C., more preferably about 30° C. to about 80° C., and most preferablyabout 37° C. to about 75° C. The temperature for the transcription stepscan be lower than the temperature(s) for the preceding steps. Thetemperature of the transcription steps can be in the range of preferablyabout 25° C. to about 85° C., more preferably about 30° C. to about 75°C., and most preferably about 37° C. to about 70° C.

Nucleotide and/or nucleotide analogs, such as deoxyribonucleosidetriphosphates, that can be employed for synthesis of the primerextension products in the methods of the invention are provided in theamount of from preferably about 50 to about 2500 μM, more preferablyabout 100 to about 2000 μM, even more preferably about 200 to about 1700μM, and most preferably about 250 to about 1500 μM. Nucleotides and/oranalogs, such as ribonucleoside triphosphates, that can be employed forsynthesis of the RNA transcripts in the methods of the invention areprovided in the amount of from preferably about 0.25 to about 6 mM, morepreferably about 0.5 to about 5 mM, even more preferably about 0.75 toabout 4 mM, and most preferably about 1 to about 3 mM.

The oligonucleotide components of the amplification reactions of theinvention are generally in excess of the number of target nucleic acidsequence to be amplified. They can be provided at about or at leastabout any of the following: 10, 10², 10⁴, 10⁶, 10⁸, 10¹⁰, 10¹² times theamount of target nucleic acid. Primers and PTO can each be provided atabout or at least about any of the following concentrations: 50 nM, 100nM, 500 nM, 1000 nM, 2500 nM, 5000 nM.

In one embodiment, the foregoing components are added simultaneously atthe initiation of the amplification process. In another embodiment,components are added in any order prior to or after appropriatetimepoints during the amplification process, as required and/orpermitted by the amplification reaction. Such timepoints, some of whichare noted below, can be readily identified by a person of skill in theart. The enzymes used for nucleic acid amplification according to themethods of the invention can be added to the reaction mixture eitherprior to the target nucleic acid denaturation step, following thedenaturation step, or following hybridization of the primer to thetarget RNA, as determined by their thermal stability and/or otherconsiderations known to the person of skill in the art. The first strandcDNA (first primer extension product) and the second strand cDNA (secondprimer extension product) synthesis reactions can be performedconsecutively, followed by the amplification steps (for example, bindingof propromoter polynucleotide and transcription). In these embodiments,the reaction conditions and components may be varied between thedifferent reactions.

The amplification reactions can be stopped at various timepoints, andresumed at a later time. Said timepoints can be readily identified by aperson of skill in the art. One timepoint is at the end of first strandcDNA synthesis. Another timepoint is at the end of second strand cDNAsynthesis. Methods for stopping the reactions are known in the art,including, for example, cooling the reaction mixture to a temperaturethat inhibits enzyme activity or heating the reaction mixture to atemperature that destroys an enzyme. Methods for resuming the reactionsare also known in the art, including, for example, raising thetemperature of the reaction mixture to a temperature that permits enzymeactivity or replenishing a destroyed (depleted) enzyme. In someembodiments, one or more of the components of the reactions isreplenished prior to, at, or following the resumption of the reactions.Alternatively, the reaction can be allowed to proceed (i.e., from startto finish) without interruption.

The reaction can be allowed to proceed without purification ofintermediate complexes, for example, to remove primer. Products can bepurified at various timepoints, which can be readily identified by aperson of skill in the art. One timepoint is at the end of first strandcDNA synthesis. Another timepoint is at the end of second strand cDNAsynthesis.

The detection of the amplification product is indicative of the presenceof the target sequence. Quantitative analysis is also feasible. Directand indirect detection methods (including quantitation) are well knownin the art. For example, by comparing the amount of product amplifiedfrom a test sample containing an unknown amount of a polynucleotidecontaining a target sequence to the product of amplification of areference sample that has a known quantity of a polynucleotide thatcontains the target sequence, the amount of target sequence in the testsample can be determined. The amplification methods of the invention canalso be extended to analysis of sequence alterations and sequencing ofthe target nucleic acid. Further, detection could be effected by, forexample, examination of translation products from RNA amplificationproducts.

Compositions and Kits of the Invention

The invention also provides compositions and kits used in the methodsdescribed herein. The compositions may be any component(s), reactionmixture and/or intermediate described herein, as well as any combinationthereof.

For example, the invention provides compositions comprising: (a) a firstprimer; (b) a second primer (which can be a random primer); and (c) apropromoter polynucleotide (such as a PTO). In some embodiments, thecompositions further comprises: (d) a third primer (which can be arandom primer). In some embodiments, the second primer and/or thirdprimer is a random primer. In some embodiments, the propromoterpolynucleotide ((c), above) is capable of hybridizing to the complementof the 5′ portion of the first primer.

The invention also provides compositions comprising a propromoterpolynucleotide (such as a PTO) capable of hybridizing to a 3′ portion ofa second primer extension that is complement of a 5′ portion of a firstprimer used to create first primer extension product.

The invention also provides compositions comprising (a) a first primer;(b) a second primer (which can be a random primer); and (c) a compositeprimer, wherein the composite primer comprises a 5′ RNA portion and aDNA portion. In some embodiments, the composition further comprises oneor more of the following: DNA-dependent DNA polymerase, RNA-dependentDNA polymerase, and an agent (generally an enzyme) that cleaves RNA froman RNA/DNA heteroduplex.

The invention also provides compositions comprising a propromoterpolynucleotide, wherein the propromoter is hybridizable to a sequence inthe second primer extension product comprising the complement of the 5′portion of a first prime, wherein the first primer is extended to formthe first primer extension product.

In another aspect, the invention provides complexes and/or reactionintermediates produced (present) in any of the methods described herein.Examples of such complexes are schematically depicted in FIGS. 1,2 and3. In one example, a complex of the invention is a complex comprising:(a) a second or third primer extension product; and (b) a propromoterpolynucleotide (for example, a PTO). In yet another example, theinvention provides compositions comprising a complex of (a) a thirdprimer extension product; and (b) a propromoter polynucleotide (whichcan be a PTO). In yet another example, the invention providescompositions comprising a complex of (a) a second primer extensionproduct, generated by denaturation of a hybridized first and secondprimer extension product; and (b) a composite primer hybridizable to thesecond primer extension product.

The invention also provides compositions comprising the amplificationproducts described herein. Accordingly, the invention provides apopulation of anti-sense RNA molecules which are copies of a targetsequence, which are produced by any of the methods described herein. Theinvention also provides a population of anti-sense polynucleotides(generally DNA) molecules, which are produced by any of the methodsdescribed herein.

The compositions are generally in a suitable medium, although they canbe in lyophilized form. Suitable media include, but are not limited to,aqueous media (such as pure water or buffers).

The invention provides kits for carrying out the methods of theinvention. Accordingly, a variety of kits are provided in suitablepackaging. The kits may be used for any one or more of the usesdescribed herein, and, accordingly, may contain instructions for any oneor more of the following uses: amplifying a RNA sequence of interest,sequencing, genotyping (nucleic acid mutation detection), preparation ofan immobilized nucleic acid (which can be a nucleic acid immobilized ona microarray), characterizing nucleic acids using the amplified nucleicacid products generated by the methods of the invention; gene expressionprofiling, subtractive hybridization; preparation of probes forsubtractive hybridization; and preparing libraries (which can be cDNAand/or differential hybridization libraries).

The kits of the invention comprise one or more containers comprising anycombination of the components described herein, and the following areexamples of such kits. For example, the invention provides kits thatcomprise a first primer that comprises a sequence that when introducedinto the amplification steps of the methods of the invention results ingeneration of an intermediate polynucleotide to which a propromoterpolynucleotide can hybridize. The invention also provides kits thatfurther comprise a second primer and/or a third primer, either of bothof which can be a random primer. The kits can contain furthercomponents, such as any of (a) a propromoter polynucleotide (such as aPTO); and (b) any of the enzymes described herein, such as an enzymewhich cleaves RNA from an RNA/DNA hybrid (for example, RNaseH), a DNApolymerase (RNA-dependent or DNA-dependent) or an RNA polymerase. Withrespect to compositions containing a random primer, these compositionsmay also contain a plurality of random primers (i.e., a population ofrandom primers having different sequences).

Kits may also optionally include any of one or more of the enzymesdescribed herein, as well as deoxynucleoside triphosphates and/orribonucleoside triphosphates. Kits may also include one or more suitablebuffers (as described herein). Kits useful for nucleic acid sequencingmay optionally include labeled or unlabeled nucleotide analogs that uponincorporation into a primer extension product effect termination ofnucleotide polymerization. One or more reagents in the kit can beprovided as a dry powder, usually lyophilized, including excipients,which on dissolution will provide for a reagent solution having theappropriate concentrations for performing any of the methods describedherein. Each component can be packaged in separate containers or somecomponents can be combined in one container where cross-reactivity andshelf life permit.

The kits of the invention may optionally include a set of instructions,generally written instructions, although electronic storage media (e.g.,magnetic diskette or optical disk) containing instructions are alsoacceptable, relating to the use of components of the methods of theinvention for the intended nucleic acid amplification, and/or, asappropriate, for using the amplification products for purposes such asnucleic acid sequencing and detection of sequence mutation. Theinstructions included with the kit generally include information as toreagents (whether included or not in the kit) necessary for practicingthe methods of the invention, instructions on how to use the kit, and/orappropriate reaction conditions. For example, the invention provideskits that comprise a first primer that comprises a sequence thecomplement of which is hybridizable by a propromoter polynucleotide, andinstructions for using the primer to amplify RNA. In another example,kits can further comprise a second primer and/or a third primer, andoptionally instructions for using the primers to amplify RNA. In otherexamples, the kits can contain further components, such as any of (a) apropromoter polynucleotide (such as a PTO); and (b) any of the enzymesdescribed herein, such as an enzyme which cleaves RNA from an RNA/DNAhybrid (for example, RNaseH), DNA polymerase (RNA-dependent orDNA-dependent) and RNA polymerase. In another example, a kit comprises(a) a composite primer; and (b) instructions for amplifying RNAaccording to any of the methods described herein. In some embodiments,said kit further comprises a PTO. In other embodiments, said kit furthercomprises one or more of the following components: (a) a first primer;(b) a second primer; (c) an agent (generally RNase H) capable ofcleaving RNA from RNA/DNA heteroduplexes; DNA-dependent DNA polymerase;and RNA-dependent DNA-polymerase. Any of these kits can further compriseinstructions for using the components to amplify RNA.

The component(s) of the kit may be packaged in any convenient,appropriate packaging. The components may be packaged separately, or inone or multiple combinations. Where kits are provided for practicingamplification methods of the invention, the RNA polymerase (if included)is preferably provided separately from the components used in the stepsprior to the transcription steps.

The relative amounts of the various components in the kits can be variedwidely to provide for concentrations of the reagents that substantiallyoptimize the reactions that need to occur to practice the methodsdisclosed herein and/or to further optimize the sensitivity of anyassay.

The invention also provides systems for effecting the methods describedherein. These systems comprise various combinations of the componentsdiscussed above. For example, the invention provides systems foramplifying a target RNA, comprising: (a) a first primer; (b) a secondprimer (which can be a random primer); (c) an RNA-dependent DNApolymerase; (d) a DNA-dependent DNA polymerase; (e) a propromoterpolynucleotide; and (f) an enzyme that cleaves RNA from an RNA/DNAhybrid. The system may further comprise: (g) a third primer (which canbe a random primer). The system may also further comprise a compositeprimer that hybridizes to a second strand cDNA. A system generallyincludes one or more apparatuses for performing the amplificationmethods of the invention. Such apparatuses include, for example, heatingdevices (such as heating blocks or water baths) and apparatuses whicheffect automation of one or more steps of the methods described herein.

The invention also provides reaction mixtures (or compositionscomprising reaction mixtures) which contain various combinations ofcomponents described herein. An example of a reaction mixture is (a) acomplex of a first primer extension product and a second primerextension product; (b) a polynucleotide comprising a propromotersequence (for example, a PTO); and (c) RNA polymerase. Other reactionmixtures are described herein and are encompassed by the invention.

Methods Using the Amplification Methods and Compositions of theInvention

The methods and compositions of the invention can be used for a varietyof purposes. For purposes of illustration, methods of sequencing,genotyping (nucleic acid mutation detection), preparation of animmobilized nucleic acid (which can be a nucleic acid immobilized on amicroarray), and characterizing nucleic acids using the amplifiednucleic acid products generated by the methods of the invention, aredescribed. Methods of expression profiling, methods of subtractivehybridization and the preparation of probes for subtractivehybridization, and methods of preparing libraries (which can be cDNAand/or differential hybridization libraries) are also described.

Sequencing of RNA Targets Using the Methods of the Invention

The amplification methods of the invention are useful, for example, forsequencing of an RNA sequence of interest. The sequencing process iscarried out as described for the amplification methods described herein.

The amplification methods of the invention are useful, for example, forsequencing of an RNA sequence of interest. The sequencing process iscarried out by amplifying a target RNA containing the sequence ofinterest by any of the methods described herein. Addition of nucleotidesduring primer extension is analyzed using methods known in the art, forexample, incorporation of a terminator nucleotide or sequencing bysynthesis (e.g. pyrosequencing).

In embodiments wherein the end product is in the form of displaced DNAprimer extension products, in addition to the nucleotides, such asnatural deoxyribonucleotide triphosphates (dNTPs), that are used in theamplification methods, appropriate nucleotide triphosphate analogs,which may be labeled or unlabeled, that upon incorporation into a primerextension product effect termination of primer extension, may be addedto the reaction mixture. Preferably, the dNTP analogs are added after asufficient amount of reaction time has elapsed since the initiation ofthe amplification reaction such that a desired amount of second primerextension product or fragment extension product has been generated. Saidamount of the time can be determined empirically by one skilled in theart.

In embodiments wherein the end product is in the form of RNA products,sequencing can be based on premature (deliberate) termination of RNAtranscription. The inclusion of rNTP analogs, which may be labeled orunlabeled, that upon incorporation into an RNA transcript effectstermination of rNTP polymerization in the reaction mixture, will resultin production of truncated RNA products, which result from blocking ofthe RNA polymerase at sites of incorporation of the analogs.

Suitable analogs (dNTP and rNTP) include those commonly used in othersequencing methods and are well known in the art. Examples of dNTPanalogs include dideoxyribonucleotides. Examples of rNTP analogs (suchas RNA polymerase terminators) include 3′-dNTP. Sasaki et al.,Biochemistry (1998) 95:3455-3460. These analogs may be labeled, forexample, with fluorochromes or radioisotopes. The labels may also belabels which are suitable for mass spectroscopy. The label may also be asmall molecule which is a member of a specific binding pair, and can bedetected following binding of the other member of the specific bindingpair, such as biotin and streptavidin, respectively, with the lastmember of the binding pair conjugated to an enzyme that catalyzes thegeneration of a detectable signal that could be detected by methods suchas colorimetry, fluorometry or chemiluminescence. All of the aboveexamples are well known in the art. These are incorporated into theprimer extension product or RNA transcripts by the polymerase and serveto stop further extension along a template sequence. The resultingtruncated polymerization products are labeled. The accumulated truncatedproducts vary in length, according to the site of incorporation of eachof the analogs, which represent the various sequence locations of acomplementary nucleotide on the template sequence.

Analysis of the reaction products for elucidation of sequenceinformation can be carried out using any of various methods known in theart. Such methods include gel electrophoresis and detection of thelabeled bands using appropriate scanner, sequencing gel electrophoresisand detection of the radiolabeled band directly by phosphorescence suchas Molecular Dynamics reader, capillary electrophoresis adapted with adetector specific for the labels used in the reaction, and the like. Thelabel can also be a ligand for a binding protein which is used fordetection of the label in combination with an enzyme conjugated to thebinding protein, such as biotin-labeled chain terminator andstreptavidin conjugated to an enzyme. The label is detected by theenzymatic activity of the enzyme, which generates a detectable signal.As with other sequencing methods known in the art, the sequencingreactions for the various nucleotide types (A, C, G, T or U) are carriedout either in a single reaction vessel, or in separate reaction vessels(each representing one of the various nucleotide types). The choice ofmethod to be used is dependent on practical considerations readilyapparent to one skilled in the art, such as the nucleotide triphosphateanalogs and/or label used. Thus, for example, when each of the analogsis differentially labeled, the sequencing reaction can be carried out ina single vessel. The considerations for choice of reagent and reactionconditions for optimal performance of sequencing analysis according tothe methods of the invention are similar to those for other previouslydescribed sequencing methods. The reagent and reaction conditions shouldbe as described above for the nucleic acid amplification methods of theinvention.

Mutation Detection, Including Mutation Detection Based on SingleStranded Conformation Polymorphism Utilizing the Amplification Methodsof the Invention

The polynucleotide (generally, RNA and DNA) amplification productsgenerated according to the methods of the invention are also suitablefor analysis for the detection of any alteration in the target nucleicacid sequence, as compared to a reference nucleic acid sequence which isidentical to the target nucleic acid sequence other than the sequencealteration. The sequence alterations may be sequence alterations presentin the genomic sequence or may be sequence alterations which are notreflected in the genomic DNA sequences, for example, alterations due topost transcriptional alterations, and/or mRNA processing, includingsplice variants.

The RNA and DNA products of the amplification methods are suitable forsingle stranded conformation polymorphism (rSSCP) based mutationdetection. The amplification methods of the invention can be directlylinked to appropriate means for detecting single stranded conformationpolymorphism, such as an electrophoretic separation method for theidentification of specific mobility pattern of the single stranded RNAor DNA products for the elucidation of the presence of specific sequencefeature(s), and/or the presence of any difference in a test nucleic acidas compared to a reference nucleic acid.

Methods based on gel electrophoresis or capillary electrophoresis can beused for the detection and analysis of the various single strandedconformational isomers. Alternatively, it is also likely that cleavageof the single stranded RNA product using nucleases which recognizesequence dependent secondary structures may be useful for thedetermination of sequence specific conformation polymorphism. Suchnucleases are known in the art, such as the Cleavase assay (Third Wave).The electrophoretic methods are potentially more suitable for highthroughput mutation, or genotyping, detection methods.

The determination of sequence specific electrophoretic pattern for agiven nucleic acid sequence is useful for, for example, the detection ofspecific alleles of a test sequence. Furthermore, it is expected that anelectrophoretic mobility pattern for the various alleles could be welldifferentiated, thus allowing the detection of two alleles in a nucleicacid sample from a single individual, as required for heterozygousgenotype, or multiple alleles. Any alteration in the test nucleic acidsequence, such as base substitution, insertions or deletion, could bedetected using this method. The method is expected to be useful fordetection of specific single base polymorphism, SNP, and the discoveryof new SNPs. Thus, the invention also provides methods for detecting apolynucleotide comprising a single nucleotide polymorphism, comprising:(a) amplifying a target polynucleotide using any of the methodsdescribed herein; and (b) analyzing the amplification products forsingle stranded conformation, wherein a difference in conformation ascompared to a reference single stranded polynucleotide indicates asingle nucleotide polymorphism in the target polynucleotide, whereby apolynucleotide comprising a single nucleotide polymorphism is detected.

Other art recognized methods of analysis for the detection of anyalteration in the target nucleic acid sequence, as compared to areference nucleic acid sequence, are suitable for use with the singlestranded nucleic acid products of the amplification methods of theinvention. Such methods are well-known in the art, and include variousmethods for the detection of specific defined sequences includingmethods based on allele specific primer extension, allele specific probeligation, differential probe hybridization, and limited primerextension. See, for example, Kurn et al, U.S. Pat. No. 6,251,639 B1;U.S. Pat. Nos. 5,888,819; 6,004,744; 5,882,867; 5,854,033; 5,710,028;6,027,889; 6,004,745; 5,763,178; 5,011,769; 5,185,243; 4,876,187;5,882,867; 5,731,146; WO US88/02746; WO 99/55912; WO 92/15712; WO00/09745; WO 97/32040; WO 00/56925; and U.S. Pat. No. 5,660,988. Thus,the invention also provides methods for detection of a mutation in anRNA sequence of interest comprising a single nucleotide polymorphism,comprising: (a) amplifying a target RNA using any of the methodsdescribed herein; and (b) analyzing the amplification products forpresence of an alteration (mutation) as compared to a reference singlestranded polynucleotide.

Method of Immobilizing Single Stranded Nucleic Acids

The single stranded polynucleotide (generally RNA and DNA) products ofsome of the amplification methods of the invention are suitable forimmobilizing to a surface. The single stranded products are particularlysuitable for preparing microarrays comprising the single strandedamplification products.

Amplification products can be immobilized and/or attached to a solid orsemi-solid support or surface, which may be made, e.g., from glass,plastic (e.g., polypropylene, nylon), polyacrylamide, nitrocellulose, orother materials.

Several techniques are well-known in the art for immobilizing nucleicacids to a solid substrate such as a glass slide. One method is toincorporate modified bases or analogs that contain a moiety that iscapable of attachment to a solid substrate, such as an amine group, aderivative of an amine group or another group with a positive charge,into the amplified nucleic acids. The amplified product is thencontacted with a solid substrate, such as a glass slide, which is coatedwith an aldehyde or another reactive group which will form a covalentlink with the reactive group that is on the amplified product and becomecovalently attached to the glass slide. Microarrays comprising theamplified products can be fabricated using a Biodot (BioDot, Inc.Irvine, Calif.) spotting apparatus and aldehyde-coated glass slides (GELAssociates, Houston, Tex.). Amplification products can be spotted ontothe aldehyde-coated slides, and processed according to publishedprocedures (Schena et al., Proc. Natl. Acad. Sci. U.S.A. (1996), 93:10614-10619). Arrays can also be printed by robotics onto glass, nylon(Ramsay, G., Nature Biotechnol. (1998), 16:40-44), polypropylene(Matson, et al., Anal Biochem. (1995), 224(1):110-6), and siliconeslides (Marshall, A. and Hodgson, J., Nature Biotechnol. (1998),16:27-31). Other approaches to array assembly include finemicropipetting within electric fields (Marshall and Hodgson, supra), andspotting the polynucleotides directly onto positively coated plates.Methods such as those using amino propyl silicon surface chemistry arealso known in the art, as disclosed at www.cmt.corning.com andcmm.stanford.edu/pbrown/.

One method for making microarrays is by making high-densitypolynucleotide arrays. Techniques are known for rapid deposition ofpolynucleotides (Blanchard et al., Biosensors & Bioelectronics,11:687-690). Other methods for making microarrays, e.g., by masking(Maskos and Southern, Nuc. Acids. Res. (1992), 20:1679-1684), may alsobe used. In principle, and as noted above, any type of array, forexample, dot blots on a nylon hybridization membrane, could be used.However, as will be recognized by those skilled in the art, very smallarrays will frequently be preferred because hybridization volumes willbe smaller.

The amplified polynucleotides may be spotted as a matrix on substratescomprising paper, glass, plastic, polypropylene, nylon, polyacrylamide,nitrocellulose, silicon, optical fiber or any other suitable solid orsemi-solid (e.g., thin layer of polyacrylamide gel (Khrapko, et al., DNASequence (1991), 1:375-388) surface.

An array may be assembled as a two-dimensional matrix on a planarsubstrate or may have a three-dimensional configuration comprising pins,rods, fibers, tapes, threads, beads, particles, microtiter wells,capillaries, cylinders and any other arrangement suitable forhybridization and detection of target molecules. In one embodiment thesubstrate to which the amplification products are attached is magneticbeads or particles. In another embodiment, the solid substrate comprisesan optical fiber. In yet another embodiment, the amplification productsare dispersed in fluid phase within a capillary which, in turn, isimmobilized with respect to a solid phase.

Characterization of Nucleic Acids

The methods of the invention are particularly amenable to quantitativeanalysis, as sufficient single stranded polynucleotide (generally, DNAand RNA) products are produced which accurately reflect therepresentation of the various mRNA in the starting material. Theamplified products can be analyzed using, for example, probehybridization techniques known in the art, such as Northern blotting,and hybridizing to probe arrays. In addition, the single strandedpolynucleotide products may serve as starting material for otherstarting material for other analytical and/or quantification methodsknown in the art, such as real time PCR, quantitative TaqMan,quantitative PCR using molecular beacons, methods described in Kurn,U.S. Pat. No. 6,251,639, etc. Thus, the invention includes those furtheranalytical and/or quantification methods as applied to any of theproducts of the methods herein.

In another embodiment, the amplification methods of the invention areutilized to generate multiple copies of single stranded polynucleotide(DNA or RNA) products that are labeled by the incorporation of labelednucleotides during DNA or RNA polymerization. For example, amplificationaccording to the methods of the invention can be carried out withsuitable labeled dNTPs or rNTPs. These labeled nucleotides can bedirectly attached to a label, or can comprise a moiety which could beattached to a label. The label may be attached covalently ornon-covalently to the amplification products. Suitable labels are knownin the art, and include, for example, a ligand which is a member of aspecific binding pair which can be detected/quantified using adetectable second member of the binding pair. Thus, amplification oftotal mRNA according to the methods of the invention in the presence of,for example, Cy3-dUTP or Cy5-dUTP results in the incorporation of thesenucleotides into the amplification products.

The labeled amplified products are particularly suitable for analysis(for example, detection and/or quantification) by contacting them with,for example, microarrays (of any suitable surface, which includes glass,chips, plastic), beads, or particles, that comprise suitable probes suchas cDNA and/or oligonucleotide probes. Thus, the invention providesmethods to characterize (for example, detect and/or quantify) an RNAsequence of interest by generating labeled polynucleotide (generally,DNA or RNA) products using amplification methods of the invention, andanalyzing the labeled products. Analysis of labeled products can beperformed by, for example, hybridization of the labeled amplificationproducts to, for example, probes immobilized at, for example, specificlocations on a solid or semi-solid substrate, probes immobilized ondefined particles, or probes immobilized on blots (such as a membrane),for example arrays, which have been described above. Other methods ofanalyzing labeled products are known in the art, such as, for example,by contacting them with a solution comprising probes, followed byextraction of complexes comprising the labeled amplification productsand probes from solution. The identity of the probes providescharacterization of the sequence identity of the amplified products, andthus by extrapolation the identity of the target RNA present in asample. Hybridization of the labeled products is detectable, and theamount of specific labels that are detected is proportional to theamount of the labeled amplification products of a specific RNA sequenceof interest. This measurement is useful for, for example, measuring therelative amounts of the various RNA species in a sample, which arerelated to the relative levels of gene expression, as described herein.The amount of labeled products (as indicated by, for example, detectablesignal associated with the label) hybridized at defined locations on anarray can be indicative of the detection and/or quantification of thecorresponding target RNA species in the sample.

The labeled amplified products are particularly suitable for analysis(for example, detection and/or quantification and/or determiningpresence or absence of) by contacting them with, for example,microarrays that comprise suitable probes such as cDNA and/oroligonucleotide probes. Thus, the invention provides methods tocharacterize (for example, detect and/or quantify and/or determinepresence or absence of) an RNA sequence of interest by generatinglabeled polynucleotide (generally, RNA or DNA) products usingamplification methods of the invention, and analyzing the labeledproducts. Analysis of labeled products can be performed by, for example,hybridization of the labeled amplification products to, for example,probes immobilized at, for example, specific locations on a solid orsemi-solid substrate, probes immobilized on defined particles, or probesimmobilized on blots (such as a membrane), for example arrays, whichhave been described above. Other methods of analyzing labeled productsare known in the art, such as, for example, by contacting them with asolution comprising probes, followed by extraction of complexescomprising the labeled amplification products and probes from solution.The identity of the probes provides characterization of the sequenceidentity of the amplified products, and thus by extrapolation theidentity of the target RNA present in a sample. Hybridization of thelabeled products is detectable, and the amount of specific labels thatare detected is proportional to the amount of the labeled amplificationproducts of a specific RNA sequence of interest. This measurement isuseful for, for example, measuring the relative amounts of the variousRNA species in a sample, which are related to the relative levels ofgene expression. The amount of labeled products (as indicated by, forexample, detectable signal associated with the label) hybridized atdefined locations on an array can be indicative of the detection and/orquantification and/or presence or absence of the corresponding targetRNA species in the sample.

Determination of Gene Expression Profile

The amplification methods of the invention are particularly suitable foruse in determining the levels of expression of multiple genes in asample since the methods described herein are capable of amplifyingmultiple target RNAs in the same sample. As described above,amplification products can be detected and quantified by variousmethods, as described herein and/or known in the art. Since RNA is aproduct of gene expression, the levels of the various RNA species, suchas mRNAs, in a sample is indicative of the relative expression levels ofthe various genes (gene expression profile). Thus, determination of theamount of RNA sequences of interest present in a sample, as determinedby quantifying amplification products of the sequences, provides fordetermination of the gene expression profile of the sample source.

Accordingly, the invention provides methods of determining geneexpression profile in a sample, said method comprising: amplifyingsingle stranded product from at least one RNA sequence of interest inthe sample, using any of the methods described herein; and determiningamount of amplification products of each RNA sequence of interest,wherein each said amount is indicative of amount of each RNA sequence ofinterest in the sample, whereby the expression profile in the sample isdetermined. Generally, labeled products are generated. In oneembodiment, the target RNA is mRNA. It is understood that amount ofamplification product may be determined using quantitative and/orqualitative methods. Determining amount of amplification productincludes determining whether amplification product is present or absent.Thus, an expression profile can includes information about presence orabsence of one or more RNA sequence of interest. “Absent” or “absence”of product, and “lack of detection of product” as used herein includesinsignificant, or de minimus levels.

The methods of expression profiling are useful in a wide variety ofmolecular diagnostic, and especially in the study of gene expression inessentially any mammalian cell (including a single cell) or cellpopulation. A cell or cell population (e.g. a tissue) may be from, forexample, blood, brain, spleen, bone, heart, vascular, lung, kidney,pituitary, endocrine gland, embryonic cells, tumors, or the like.Expression profiling is also useful for comparing a control (normal)sample to a test sample, including test samples collected at differenttimes, including before, after, and/or during development, a treatment,and the like.

Method of Preparing a Library

The single stranded polynucleotides (generally DNA and RNA) products ofthe methods of the invention are useful in preparing libraries,including cDNA libraries and subtractive hybridization libraries. Usingthe methods of the invention, libraries may be prepared from limitedamount of starting material, for example, mRNA extracted from limitedamount of tissue or even single cells. Accordingly, in one aspect, themethods of the invention provides preparing a library from the singlestranded DNA or RNA products of the invention. In still another aspect,the invention provides methods for making a library, said methodcomprising: preparing a subtractive hybridization probe using any of themethods described herein.

Accordingly, in one aspect, the methods of the invention providespreparing a library from the single stranded DNA or RNA products of theinvention. In some embodiments, the library is a cDNA library.

Methods of Subtractive Hybridization

The amplification methods of the invention are particularly suitable foruse in subtractive hybridization methods, since the methods describedherein are capable of amplifying multiple target RNAs in the samesample, and the methods of the invention are suitable for producinglarge amounts of single stranded anti-sense DNA and RNA product suitablefor use as “driver” in subtractive hybridization. For example, twonucleic acid populations, one sense and one antisense, can be allowed tomix together with one population present in molar excess (“driver”).Sequence present in both populations will form hybrids, while sequencespresent in only one population remain single-stranded. Thereafter,various well known techniques are used to separate the unhybridizedmolecules representing differentially expressed sequences. See, e.g.,Hamson et al., U.S. Pat. No. 5,589,339; Van Gelder, U.S. Pat. No.6,291,170;

Accordingly, the invention provides methods for performing subtractivehybridization, said methods comprising: (a) preparing multiple copies(generally, DNA) of the complement of at least one RNA sequences ofinterest from a first RNA population using any of the amplificationmethods described herein; and (b) hybridizing the multiple copies to asecond mRNA population, whereby a subpopulation of the second mRNApopulation forms a complex with a nucleotide DNA copy. The inventionalso provides methods for performing subtractive hybridization, saidmethods comprising: hybridizing multiple copies of the complement of atleast one RNA sequences of interest from a first RNA population usingany of the amplification methods described herein to a second mRNApopulation, whereby a subpopulation of the second mRNA population formsa complex with a copy. In some embodiments, “driver” single strandedanti-sense DNA product of the methods of the invention is combined withtester (sense) mRNA species. In another aspect, the invention providesmethods of differential amplification in which single stranded driver(antisense) DNA sequences that hybridize with tester mRNA sequence aresubjected to cleavage by an agent that cleaves RNA present in a DNA/RNAhybrid, such as RNase H. Cleavage of the mRNA results in the inabilityto generate single stranded DNA product from the test mRNA strands.Conversely, non-cleaved tester (i.e., tester mRNA that did not hybridizeto driver DNA molecules) may serve as a substrate for subsequentamplification. Amplified differentially expressed products have manyuses, including as a differential expression probe, to producedifferential expression libraries Accordingly, in another aspect, theinvention provides methods comprising hybridizing multiplepolynucleotide (generally, DNA) copies of the complement of at least oneRNA sequences of interest from a first RNA population using any of theamplification methods described herein to a second mRNA population,whereby a subpopulation of the second mRNA population forms a complexwith a DNA copy; (b) cleaving RNA in the complex of step (a) with anenzyme that cleaves RNA from an RNA/DNA hybrid; and (c) amplifying anunhybridized subpopulation of the second mRNA population, wherebymultiple copies of single stranded polynucleotide (generally, DNA)complementary to the unhybridized subpopulation of the second mRNApopulation are generated.

The following Examples are provided to illustrate, but not limit, theinvention.

EXAMPLES Example 1 Amplification of Total mRNA

0.1 μg of total mRNA is combined with primer 1 (provided at aconcentration of from 0.1 to 1 μM), PTO oligonucleotide (provided at aconcentration of from 0.1 to 1 μM), primer 2 (provided at aconcentration of from 0.1 to 1 μM) in a total volume of about 10 μ in abuffer containing 40 mM Tris, pH 8.5, 5 mM DTT, 12 MM MgCl₂, 70 mM KCl,108.8 μg/ml BSA; 1 mM of each dNTP, 2 mM each of rATP, rUTP, rCTP, 1.5mM rGTP, and 0.5 mM rITP). The mixture is incubated at 65° C. for 2minutes, and cooled down to 37° C. (or 42° C.). 10 μl of an enzymemixture containing T7 RNA polymerase (40 to 80 U), MMLV reversetranscriptase (10 to 30 U), and RNase H (1 to 3 U), is added to thereaction mixture, and the mixture is further incubated for 0.5 to 3hours.

Aliquots of the reaction mixture are analyzed by gel electrophoresis (5to 20% PAGE, Novex) for generation of amplification products.

Primer 1 sequence:

-   GACGGATGCGGTCTTTTTTTTN (SEQ NO:1)-   “N” denotes a degenerate nucleotide (i.e., it can be A, T, C or G).    Primer 2:-   Random hexamer    PTO.-   ggAATTCTAATACgACTCACTATAgggAgAgCGACGGATGCGGTCT-biotin (SEQ ID NO:2)-   wherein bold letters denote the sequence complementary to the 3′-end    of the second primer extension product.    Primer 3:-   Primer 3 is the same as primer 1.

Example 2 Amplification of Specific mRNA

The experiment of Example 1 is performed, except that primer 2 issubstituted with a primer specific for a sequence of a defined mRNAspecies to be amplified. For example, the amplification of a sequence ofGAPDH mRNA is carried out with a primer that hybridizes to a site on thefirst cDNA strand (first primer extension product) generated from GAPDHmRNA.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be apparent to those skilled in the art thatcertain changes and modifications may be practiced. Therefore, thedescriptions and examples should not be construed as limiting the scopeof the invention, which is delineated by the appended claims.

1. A method of generating multiple copies of the complementary sequenceof an RNA sequence of interest, said method comprising the steps of: (a)extending a first primer hybridized to a target RNA with anRNA-dependent DNA polymerase, whereby a complex comprising a firstprimer extension product and the target RNA is produced; (b) cleavingRNA in the complex of step (a) with an enzyme that cleaves RNA from anRNA/DNA hybrid; (c) extending a second primer hybridized to the firstprimer extension product with a DNA-dependent DNA polymerase, whereby acomplex comprising the first primer extension product and a secondprimer extension product is produced; (d) denaturing the complex of step(c); and (e) hybridizing to the second primer extension product apropromoter polynucleotide comprising a propromoter and a region whichhybridizes to the second primer extension product under conditions whichallow transcription to occur by RNA polymerase, such that RNAtranscripts are produced comprising sequences complementary to thetarget RNA; whereby multiple copies of the complementary sequence of theRNA sequence of interest are generated.
 2. A method of generatingmultiple copies of the complementary sequence of an RNA sequence ofinterest, said method comprising the steps of: (a) extending a firstprimer hybridized to a target RNA with an RNA-dependent DNA polymerase,whereby a complex comprising a first primer extension product and thetarget RNA is produced; (b) cleaving RNA in the complex of step (a) withan enzyme that cleaves RNA from an RNA/DNA hybrid; (c) extending asecond primer hybridized to the first primer extension product with aDNA-dependent DNA polymerase, whereby a complex comprising the firstprimer extension product and a second primer extension product isproduced; (d) denaturing the complex of step (c); (e) hybridizing to thesecond primer extension product a first propromoter polynucleotidecomprising a propromoter and a region which hybridizes to the secondprimer extension product under conditions which allow transcription tooccur by RNA polymerase, such that RNA transcripts are producedcomprising sequences complementary to the target RNA; (f) extending anexponential amplification primer hybridized to said RNA transcripts withan RNA-dependent DNA polymerase, whereby a complex comprising anexponential amplification primer extension product and an RNA transcriptis produced; (g) cleaving RNA in the complex of step (f) with an enzymethat cleaves RNA from an RNA/DNA hybrid; (h) hybridizing a secondpropromoter polynucleotide comprising a propromoter and a region whichhybridizes to a single stranded exponential amplification primerextension product under conditions which allow transcription to occur byRNA polymerase, such that RNA transcripts are produced comprisingsequences complementary to the target RNA; (i) optionally repeatingsteps (f) to (h); whereby multiple copies of the complementary sequenceof the RNA sequence of interest are produced.
 3. A method of generatingmultiple copies of the complementary sequence of an RNA sequence ofinterest comprising incubating a reaction mixture, said reaction mixturecomprising: (a) a single stranded second primer extension productresulting from step (d) of claim 1; (b) a propromoter polynucleotidecomprising a propromoter and a region which is hybridizable to thesingle stranded second primer extension product; and an RNA polymerase;wherein the incubation is under conditions that permit propromoterpolynucleotide hybridization and RNA transcription, whereby multiplecopies of the complementary sequence of the RNA sequence of interest aregenerated.
 4. A method of generating multiple copies of thecomplementary sequence of an RNA sequence of interest comprisingincubating a reaction mixture, said reaction mixture comprising: (a) asingle stranded second primer extension product resulting from step (d)of claim 1; (b) an exponential amplification primer comprising asequence hybridizable to an RNA transcript comprising a sequencecomplementary to the target RNA; (c) a first propromoter polynucleotidecomprising a propromoter and a region which is hybridizable to a singlestranded second primer extension product; (d) a second propromoterpolynueleotide comprising a propromoter and a region which ishybridizable to a single stranded exponential amplification extensionproduct; (e) an enzyme that cleaves RNA from an RNA/DNA hybrid; and (f)an RNA polymerase; wherein the incubation is under conditions thatpermit primer extension, RNA cleavage, propromoter polynucleotidehybridization and RNA transcription, whereby multiple copies of thecomplementary sequence of the RNA sequence of interest are generated. 5.A method of generating multiple copies of the complementary sequence ofan RNA sequence of interest, said method comprising incubating areaction mixture, said reaction mixture comprising: (a) an RNAtranscript from step (e) of claim 1; (b) an exponential amplificationprimer comprising a sequence hybridizable to the RNA transcript; (c) apropromoter polynucleotide comprising a propromoter and a region whichis hybridizable to a single stranded exponential amplification primerextension product; (d) an enzyme that cleaves RNA from an RNA/DNAhybrid; and (e) an RNA polymerase; wherein the incubation is underconditions that permit primer extension, RNA cleavage, propromoterpolynucleotide hybridization and RNA transcription, whereby multiplecopies of the complementary sequence of the RNA sequence of interest aregenerated.
 6. A method of generating multiple copies of thecomplementary sequence of an RNA sequence of interest, said methodcomprising incubating a reaction mixture, said reaction mixturecomprising: (a) a target RNA; (b) a first primer comprising a sequencethat is hybridizable to the target RNA; (c) a second primer comprising asequence hybridizable to an extension product of the first primer; (d) apropromoter polynucleotide comprising a propromoter and a region whichis hybridizable to a single stranded second primer extension product;(e) an RNA-dependent DNA polymerase; (f) a DNA-dependent DNA polymerase;(g) an RNA polymerase; and (h) an enzyme that cleaves RNA from anRNA/DNA hybrid; wherein the incubation is under conditions that permitprimer hybridization, primer extension, RNA cleavage, propromoterpolynucleotide hybridization, and RNA transcription, whereby multiplecopies of the complementary sequence of the RNA sequence of interest aregenerated.
 7. A method of generating multiple copies of thecomplementary sequence of an RNA sequence of interest, said methodcomprising incubating a reaction mixture, said reaction mixturecomprising: (a) a target RNA; (b) a first primer comprising a sequencethat is hybridizable to the target RNA; (c) a second primer comprising asequence hybridizable to an extension product of the first primer; (d)an exponential amplification primer comprising a sequence hybridizableto an RNA transcript comprising a sequence complementary to the targetRNA; (e) a first propromoter polynucleotide comprising a propromoter anda region which is hybridizable to a single stranded second primerextension product; (f) a second propromoter polynucleotide comprising apropromoter and a region which is hybridizable to a single strandedexponential amplification primer extension product; (g) an RNA-dependentDNA polymerase; (h) a DNA-dependent DNA polymerase; (i) an RNApolymerase; and (j) an enzyme that cleaves RNA from an RNA/DNA hybrid;wherein the incubation is conditions that permit primer hybridization,primer extension, RNA cleavage, propromoter polynucleotidehybridization, and RNA transcription, whereby multiple copies of thecomplementary sequence of the RNA sequence of interest are generated. 8.A method of generating multiple copies of the complementary sequence ofan RNA sequence of interest, said method comprising incubating areaction mixture, said reaction mixture comprising: (a) a target RNA;(b) a first primer comprising a sequence that is hybridizable to thetarget RNA; (c) a propromoter polynucleotide comprising a propromoterand a region which is hybridizable to a single stranded second strandcDNA, said second strand cDNA complementary to an extension product ofthe first primer; (d) an RNA-dependent DNA polymerase; (e) aDNA-dependent DNA polymerase; (f) an RNA polymerase; and (g) an enzymethat cleaves RNA from an RNA/DNA hybrid, wherein the incubation is underconditions that permit primer hybridization, primer extension, RNAcleavage, propromoter polynucleotide hybridization, and RNAtranscription, whereby multiple copies of the complementary sequence ofthe RNA sequence of interest are generated.
 9. A method of generatingmultiple copies of the complementary sequence of an RNA sequence ofinterest, said method comprising incubating a reaction mixture, saidreaction mixture comprising: (a) a target RNA; (b) a first primercomprising a sequence that is hybridizable to the target RNA; (c) anexponential amplification primer comprising a sequence hybridizable toan RNA transcript comprising a sequence complementary to the target RNA;(d) a propromoter polynucleotide comprising a propromoter and a regionwhich is hybridizable to a single stranded second strand cDNA, saidsecond strand cDNA complementary to an extension product of the firstprimer; (e) a propromoter polynucleotide comprising a propromoter and aregion which is hybridizable to a single stranded exponentialamplification primer extension product; (f) an RNA-dependent DNApolymerase; (g) a DNA-dependent DNA polymerase; (h) an RNA polymerase;and (i) an enzyme that cleaves RNA from an RNA/DNA hybrid, wherein theincubation is conditions that permit primer hybridization, primerextension, RNA cleavage, propromoter polynucleotide hybridization, andRNA transcription, whereby multiple copies of the complementary sequenceof the RNA sequence of interest are generated.
 10. A method ofgenerating multiple copies of the complementary sequence of an RNAsequence of interest, said method comprising the steps of: (a)synthesizing a first primer extension product by extending a firstprimer hybridized to a target RNA, whereby a complex comprising thefirst primer extension product and the target RNA is produced; (b)synthesizing a second primer extension product by extending a secondprimer hybridized to the first primer extension product, whereby acomplex comprising the first primer extension product and the secondprimer extension product is produced; (c) denaturing the complex of step(b); (d) hybridizing to the second primer extension product apropromoter polynucleotide comprising a propromoter and a region whichhybridizes to the second primer extension product; and (e) synthesizingRNA transcripts comprising sequences complementary to the target RNA,whereby multiple copies of the complementary sequence of the RNAsequence of interest are generated.
 11. A method of generating multiplecopies of the complementary sequence of an RNA sequence of interest,said method comprising the steps of: (a) synthesizing a first primerextension product by extending a first primer hybridized to a targetRNA, whereby a complex comprising the first primer extension product andthe target RNA is produced; (b) synthesizing a second primer extensionproduct by extending a second primer hybridized to the first primerextension product, whereby a complex comprising the first primerextension product and the second primer extension product is produced;(c) denaturing the complex of step (b); (d) hybridizing to the secondprimer extension product a first propromoter polynucleotide comprising apropromoter and a region which hybridizes to the second primer extensionproduct; (e) synthesizing RNA transcripts comprising sequencescomplementary to the target RNA, such that RNA transcripts are producedcomprising sequences complementary to the target RNA; (f) synthesizing asingle stranded exponential amplification primer extension product byextending an exponential amplification primer hybridized to said RNAtranscripts, whereby a complex comprising an exponential amplificationprimer extension product and an RNA transcript is produced; (g)synthesizing RNA transcripts comprising sequences complementary to thetarget RNA by hybridizing a second propromoter polynucleotide comprisinga propromoter and a region which hybridizes to the single strandedexponential amplification primer extension product under conditionswhich allow transcription to occur by RNA polymerase, such that RNAtranscripts are produced comprising sequences complementary to thetarget RNA; and (h) optionally repeating steps (f) to (g); wherebymultiple copies of the complementary sequence of the RNA sequence ofinterest are produced.
 12. The method of claim 2, wherein the firstprimer comprises a 5′ portion that is not hybridizable to the targetRNA.
 13. The method of claim 12, wherein said 5′ portion comprises asequence the complement of which is hybridizable by the propromoterpolynucleotide in step (e) or (h).
 14. The method of claim 1 or 2,wherein the target RNA is mRNA.
 15. The method of claim 1 or 2, whereinthe first primer comprises a poly-T sequence.
 16. The method of claim 2,wherein the first primer comprises a 5′ portion that is not hybridizableto the target RNA, and wherein said 5′ portion comprises a sequence thecomplement of which is hybridizable by the propromoter polynucleotide instep (e) or (h).
 17. The method of claim 2, wherein the second primerand the exponential amplification primer are the same.
 18. The method ofclaim 2, wherein the second primer and the exponential amplificationprimer are different.
 19. The method of claim 18, wherein the secondprimer and the exponential amplification primer hybridize to differentcomplementary sequences.
 20. The method of claim 1 or 2, wherein atleast one type of rNTP used is a labeled rNTP, whereby labeled productsare generated.
 21. The method of claim 1 or 2, wherein the first primeris a random primer.
 22. The method of claim 1 or 2, wherein the secondprimer comprises DNA.
 23. The method of claim 1 or 2, wherein the secondprimer comprises a fragment of the target RNA hybridized to the primerextension product, said fragment generated by cleaving RNA in thecomplex of step (b) with an enzyme that cleaves RNA from an RNA/DNAhybrid.
 24. The method of claims 1 or 2, wherein said method comprisesgenerating multiple copies of two or more different sequences ofinterest.
 25. The method of claim 24, wherein said method comprises atleast two different first primers.
 26. The method of any of claims 6, 7,8, and 9, wherein the first primer comprises a 5′ sequence that is nothybridizable to the target RNA.
 27. The method of claim 26, wherein the5′ sequence comprises a sequence the complement of which is hybridizableby a propromoter polynucleotide.
 28. The method of any of claims 6, 7,8, and 9, wherein the first primer comprises a poly-T sequence; and thetarget RNA is mRNA.
 29. The method of claim 7, wherein the second primerand the exponential amplification primer are the same.
 30. The method ofclaim 7, wherein the second primer and the exponential amplificationprimer are different.
 31. The method of claim 30, wherein the secondprimer and the exponential amplification primer hybridize to differentcomplementary sequences.
 32. The method of claims 1, 2, 4, 5, 6, 7, 8and 9, wherein the enzyme that cleaves RNA is RNase H.
 33. The method ofany of claims 1, 2, 6, 7, 8, and 9, wherein the RNA-dependent DNApolymerase and DNA-dependent DNA polymerase are one enzyme.
 34. Themethod of any of claims 1, 2, 6, 7, 8, and 9, wherein the RNA-dependentDNA polymerase and enzyme that cleaves RNA from an RNA/DNA hybrid arethe same enzyme.
 35. The method of any of claims 1, 2, 6, 7, 8, and 9,wherein the DNA-dependent DNA polymerase and enzyme that cleaves RNAfrom an RNA/DNA hybrid are the same enzyme.
 36. The method of any ofclaims 1, 2, 6, 7, 8, and 9, wherein the DNA-dependent DNA polymerase,the RNA-dependent DNA polymerase and the enzyme that cleaves RNA from anRNA/DNA hybrid are the same enzyme.
 37. The method of any of claims 1,6, and 8, wherein the propromoter polynucleotide comprises a region atthe 3′ end which hybridizes to the second primer extension product,whereby DNA polymerase extension of the second primer extension productproduces a double stranded promoter from which transcription occurs. 38.The method of claim 37, wherein the propromoter polynucleotide is apropromoter template oligonucleotide.
 39. The method of any of claims 2,7, and 9, wherein the propromoter polynucleotide comprises a region atthe 3′ end which hybridizes to the exponential amplification primerextension product, whereby DNA polymerase extension of the second primerextension product produces a double stranded promoter from whichtranscription occurs.
 40. The method of claim 39, wherein thepropromoter polynucleotide is a propromoter template oligonucleotide.41. The method of any of claims 6, 7, 8, and 9, wherein at least onetype of rNTP used is a labeled rNTP, whereby labeled products aregenerated.
 42. A method of sequencing an RNA sequence of interest, saidmethod comprising analyzing amplification products to determinesequence, said amplification products produced by the method of any ofclaims 1, 6, 7, and 10 in the presence of a mixture of rNTPs and rNTPanalogs such that transcription is terminated upon incorporation of anrNTP analog.
 43. The method of claim 42, wherein the target RNA is mRNA.44. A method of sequencing an RNA sequence of interest, said methodcomprising analyzing amplification products to determine sequence, saidamplification products produced by the method of any of claims 2, 7, 9,and 11, wherein RNA transcripts generated from the second primerextension product are amplified in the presence of a mixture of rNTPsand rNTP analogs such that transcription is terminated uponincorporation of an rNTP analog.
 45. The method of claim 44, wherein thetarget RNA is mRNA.
 46. A method of detecting a mutation in a targetRNA, comprising analyzing sequences of amplification products for thepresence of a mutation as compared to a reference polynucleotidesequence, said amplification products produced by the method of any ofclaims 1, 2, 6, 7, 8, 9, and
 10. 47. The method of claim 46, wherein thetarget RNA is mRNA.
 48. A method of detecting a mutation in a target RNAby single stranded conformation polymorphism, comprising analyzingamplification products for single stranded conformation, saidamplification products produced by the method of any of claims 1, 2, 6,7, 8, 9, 10, and 11, wherein a difference in conformation as compared toa reference single stranded polynucleotide indicates a mutation in thetarget polynucleotide.
 49. The method of claim 48, wherein the targetRNA is mRNA.
 50. A method of producing a nucleic acid immobilized to asubstrate comprising immobilizing amplification products on a substrate,said amplification products produced by the method of any of claims 1,2, 6, 7, 8, 9, 10, and
 11. 51. The method of claim 50, wherein thetarget RNA is mRNA.
 52. The method of claim 50, wherein the substrate isa microarray.
 53. A method of characterizing an RNA sequence ofinterest, comprising analyzing labeled RNA products, said labeled RNAproducts produced by amplifying a target RNA by the method of claim 20.54. The method of claim 53, wherein analyzing comprises contacting thelabeled RNA products with at least one probe sequence.
 55. The method ofclaim 54, wherein the at least one probe sequence is provided as amicroarray.
 56. The method of claim 55, wherein the microarray comprisesat least one probe immobilized on a substrate fabricated from a materialselected from the group consisting of paper, ceramic, glass, plastic,polypropylene, polystyrene, nylon, polyacrylamide, nitrocellulose,silicon, and optical fiber.
 57. The method of claim 56, wherein theprobe is immobilized on the substrate in a two-dimensional configurationor a three-dimensional configuration comprising pins, rods, fibers,tapes, threads, beads, particles, microtiter wells, capillaries, andcylinders.
 58. The method of claim 53, wherein the target RNA is mRNA.59. The method of claim 53, wherein analyzing the labeled RNA productscomprises determining amount of said products, whereby the amount of theRNA sequence of interest present in a sample is quantified.
 60. A methodof characterizing an RNA sequence of interest, comprising analyzinglabeled RNA products, said labeled RNA products produced by amplifying atarget RNA by the method of claim
 41. 61. The method of claim 60,wherein analyzing comprises contacting the labeled DNA products with atleast one probe.
 62. The method of claim 61, wherein the at least oneprobe is provided as a microarray.
 63. The method of claim 62, whereinthe microarray comprises at least one probe immobilized on a substratefabricated from a material selected from the group consisting of paper,glass, plastic, polypropylene, nylon, polyacrylamide, nitrocellulose,silicon, and optical fiber.
 64. The method of claim 63, wherein theprobe is immobilized on the substrate in a two-dimensional configurationor a three-dimensional configuration comprising pins, rods, fibers,tapes, threads, beads, particles, microtiter wells, capillaries, andcylinders.
 65. The method of claim 60, wherein the target RNA is mRNA.66. The method of claim 60, wherein analyzing the labeled RNA productscomprises determining amount of said products, whereby the amount of theRNA sequence of interest present in a sample is quantified.
 67. A methodof determining gene expression profile in a sample, said methodcomprising determining amounts of amplification products of each of atleast two RNA sequences of interest, said amplification productsamplified by the method of any of claims 1, 2, 6, 7, 8, 9, 10, and 11,wherein each said amount is indicative of amount of each RNA sequence ofinterest in the sample, whereby the gene expression profile in thesample is determined.
 68. The method of claim 67, wherein each targetRNA is mRNA.
 69. A method of preparing a library, said methodcomprising: preparing a library from amplified single stranded DNA orRNA product, said amplified single stranded DNA or RNA product amplifiedby amplifying at least two RNA sequences of interest using the method ofany one of claims 1, 2, 6, 7, 8, 9, 10, and
 11. 70. The method of claim69, wherein the first primer is a random primer.
 71. The method of claim69, wherein the first primer comprises a poly-T portion.
 72. A methodfor generating multiple copies of a sequence complementary to an RNAsequence of interest comprising: (a) hybridizing to a single strandedsecond primer extension product, a propromoter polynucleotide comprisinga propromoter and a region which hybridizes to the second primerextension product; and (b) synthesizing RNA transcripts comprisingsequences complementary to the target RNA whereby multiple copies of thesequence complementary to the RNA sequence of interest are generated,wherein the second primer extension product is prepared by synthesizinga first primer extension product by extending a first primer hybridizedto the RNA sequence of interest, whereby a first complex comprising thefirst primer extension product and the RNA sequence of interest isproduced, then synthesizing a second primer extension product byextending a second primer hybridized to the first primer extensionproduct, whereby a second complex comprising the first primer extensionproduct and the second primer extension product is produced, anddenaturing the second complex.
 73. A method of performing subtractivehybridization, said method comprising: hybridizing multiple DNA copiesof the complement of one or more RNA sequences of interest from a firstRNA population to a second mRNA population, said multiple DNA copiesprepared using the method of claim 72 whereby a subpopulation of thesecond mRNA population forms a complex with the DNA copies.
 74. A methodof performing subtractive hybridization, said method comprising: (a)hybridizing multiple DNA copies of the complement of at least two RNAsequences of interest to a second mRNA population, said multiple DNAcopies prepared from a first RNA population using the method of claim72; whereby a subpopulation of the second mRNA population forms acomplex with a DNA copy; (b) cleaving RNA in the complex of step (a)with an enzyme that cleaves RNA from an RNA/DNA hybrid; and (c)amplifying the unhybridized subpopulation of the second mRNA population,whereby multiple copies of single stranded DNA complementary to theunhybridized subpopulation of the second mRNA population are generated.75. The method of claim 1, wherein the first primer comprises a 5′portion that is not hybridizable to the target RNA.
 76. The method ofclaim 10 or 11, wherein the first primer comprises a 5′ portion that isnot hybridizable to the target RNA.
 77. The method of claim 76, whereinsaid 5′ portion comprises a sequence the complement of which ishybridizable to one or more of the propromoter polynucleotides of steps(d) and (g).
 78. The method of claim 10 or 11, wherein the target RNA ismRNA.
 79. The method of claim 72, wherein the first primer comprises apoly-dT sequence.
 80. The method of claim 11, wherein the first primercomprises a 5′ portion that is not hybridizable to the target RNA, andwherein said 5′ portion comprises a sequence the complement of which ishybridizable to one or more of the propromoter polynucleotides of steps(d) and (g).
 81. The method of claim 11, wherein the second primer andthe exponential amplification primer are the same.
 82. The method ofclaim 11, wherein the second primer and the exponential amplificationprimer are different.
 83. The method of claim 82, wherein the secondprimer and the exponential amplification primer hybridize to differentcomplementary sequences.
 84. The method of claim 10 or 11, wherein thesynthesizing of RNA transcripts is carried out in the presence of atleast one type of labeled rNTP, whereby labeled products are generated.85. The method of claim 10 or 11, wherein the first primer is a randomprimer.
 86. The method of claim 10 or 11, wherein the second primercomprises DNA.
 87. The method of claim 10 or 11, wherein the secondprimer comprises a fragment of the target RNA hybridized to the primerextension product, said fragment generated by cleaving RNA in thecomplex of step (a).
 88. The method of claim 10 or 11, wherein saidmethod comprises generating multiple copies of two or more different RNAsequences of interest.
 89. The method of claim 88, wherein said methodcomprises at least two different first primers.
 90. The method of claim85, wherein the first and second propromoter polynucleotides are thesame.
 91. The method of claim 85, wherein the first and secondpropromoter polynucleotides are different.
 92. The method of claim 72,wherein the first primer comprises a 5′ portion that is not hybridizableto the RNA sequence of interest.
 93. The method of claim 72, wherein theRNA sequence of interest is mRNA.
 94. The method of claim 93, whereinthe first primer comprises a poly-dT sequence.
 95. The method of claim72, wherein the synthesis of RNA transcripts is carried out in thepresence of at least one type of labeled rNTP, whereby labeled productsare generated.
 96. The method of claim 72, wherein the first primer is arandom primer.
 97. The method of claim 72, wherein said method comprisesgenerating multiple copies of two or more different RNA sequences ofinterest.
 98. The method of claim 97, wherein said method comprises atleast two different first primers.